Peptide conjugates of microtubule-targeting agents as therapeutics

ABSTRACT

The present invention relates to peptide conjugates of microtubule-targeting agents such as maytansinoid derivatives which are useful for the treatment of diseases such as cancer.

FIELD OF THE INVENTION

The present invention relates to peptide conjugates ofmicrotubule-targeting agents such as maytansinoid derivatives which areuseful for the treatment of diseases such as cancer.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 8, 2020, isnamed 0008001SEQ.txt and is 142,925 bytes in size.

BACKGROUND OF THE INVENTION

Cancer is a group of diseases characterized by aberrant control of cellgrowth. The annual incidence of cancer is estimated to be in excess of1.6 million in the United States alone. While surgery, radiation,chemotherapy, and hormones are used to treat cancer, it remains thesecond leading cause of death in the U.S. It is estimated that about600,000 Americans will die from cancer each year.

Treatment of cancer in humans by systemic administration ofpharmaceutical agents often functions by slowing or terminating theuncontrolled replication that is a characteristic of cancer cells. Oneclass of such agents is microtubule-targeting agents. Cell divisionrequires formation of an intact mitotic spindle apparatus, composed ofmicrotubules undergoing random length changes. The random length changesof microtubules is referred to as dynamic instability. Disruption of thedynamic instability of microtubules can lead to the suppression offurther cell division. Drugs that target microtubules to suppressdynamic instability are currently used in the clinic as effectiveanticancer agents for a wide variety of cancers. See Lopus, M, CancerLett., 2011, 307(2): 113-118.

The maytansinoids, (e.g., mertansine, DM1, or DM4) are a class ofmicrotubule-targeting agents that have emerged as potential clinicalchemotherapeutics. See Lopus, M, Cancer Lett., 2011, 307(2): 113-118;and Widdison, W., J. Med. Chem. 2006, 49:4392-4408 Although DM1 has beenshown to be effective in the treatment of several types of cancer,including lymphoma and breast cancer, toxic side effects such asperipheral neuropathy have hindered the clinical development oftubulin-targeting agents such as the maytansinoids. Preferentialdelivery of maytansinoid compounds, such as DM1, to diseased tissuescould avoid these serious side effects. Thus, there is a need for moreselective delivery of maytansinoid compounds to diseased tissue.

SUMMARY

The present disclosure provides, inter alia, a compound of Formula (I):R²-L-R¹  (I)or a pharmaceutically acceptable salt thereof, wherein constituentvariables are defined herein.

The present disclosure further provides a pharmaceutical compositioncomprising a compound of the disclosure, or a pharmaceuticallyacceptable salt thereof, and at least one pharmaceutically acceptablecarrier or excipient.

The present disclosure also provides methods of treating a disease orcondition (e.g., cancer) by administering to a human or other mammal inneed of such treatment a therapeutically effective amount of a compoundof the disclosure. In some embodiments, the disease or condition ischaracterized by acidic or hypoxic diseased tissues.

The present disclosure also provides use of a compound described hereinin the manufacture of a medicament for use in therapy. The presentdisclosure also provides the compounds described herein for use intherapy.

The present disclosure also provides methods for synthesizing thecompounds of the disclosure and intermediates useful in these methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of the effect of free DM4 and Compound 5 on in vitroβ-tubulin polymerization (in terms of relative fluorescence units) atvarious concentrations.

FIG. 2 depicts the kinetic analysis of Compound 5 binding to β-tubulinin vitro as determined by Biacore surface plasmon resonance.

FIG. 3A shows a plot of the mean tumor volume in nude mice bearingHCT116 colorectal flank tumors dosed with DM4 or Compound 5.

FIG. 3B shows the percent change in body weight of nude mice bearingHCT116 colorectal flank tumors dosed with DM4 or Compound 5 relative today 0.

FIG. 4 depicts a Kaplan-Meier plot of nude mice bearing HCT116colorectal flank tumors dosed with DM4 or Compound 5.

FIG. 5A depicts the ventral view and extracted lungs of nude miceinoculated with 4T1-RFP fluorescent cells via tail vein injection andimaged 11 days after inoculation and after 3 doses of vehicle orCompound 6.

FIG. 5B depicts a graph of the fluorescent signal from extracted lungsof 4T1-RFP inoculated mice after 3 doses of vehicle or Compound 6.

DETAILED DESCRIPTION

Provided herein is a compound of Formula (I):R²-L-R¹  (I)or a pharmaceutically acceptable salt thereof, wherein:

R¹ is a peptide;

R² is a small molecule microtubule targeting moiety; and

L is a linker, which is covalently linked to moiety R¹ and R².

Provided herein is a compound of Formula (I):R²-L-R¹  (I)or a pharmaceutically acceptable salt thereof, wherein:

R¹ is a peptide having 5 to 50 amino acids;

R² is a small molecule microtubule targeting moiety; and

L is a linker, which is covalently linked to moiety R¹ and R².

Also provided herein is a compound of Formula (I):R²-L-R¹  (I)or a pharmaceutically acceptable salt thereof, wherein:

R¹ is a peptide capable of selectively delivering R²L- across a cellmembrane having an acidic or hypoxic mantle;

R² is a small molecule microtubule targeting moiety; and

L is a linker, which is covalently linked to moiety R¹ and R².

In some embodiments, R² is a maytansine-derived microtubule targetingmoiety.

Provided herein is a compound of Formula (I):R²-L-R¹  (I)or a pharmaceutically acceptable salt thereof, wherein:

R¹ is a peptide capable of selectively delivering R²L- across a cellmembrane having an acidic or hypoxic mantle;

R² is selected from the group consisting of:

and

L is a linker, which is covalently linked to moiety R¹ and R².

Provided herein is a compound of Formula (I):R²-L-R¹  (I)or a pharmaceutically acceptable salt thereof, wherein:

R¹ is a peptide capable of selectively delivering R²L- across a cellmembrane having an acidic or hypoxic mantle;

R² is selected from the group consisting of:

and

L is a linker, which is covalently linked to moiety R¹ and R².

Provided herein is a compound of Formula (I):R²-L-R¹  (I)or a pharmaceutically acceptable salt thereof, wherein:

R¹ is a peptide capable of selectively delivering R²L- across a cellmembrane having an acidic or hypoxic mantle;

R² is selected from the group consisting of:

L is a group selected from:

wherein

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each independently selected fromH, C₁₋₄ alkyl, C₁₋₄ alkenyl, C₆₋₁₀ aryl, C₃₋₁₀ cycloalkyl, 5-10 memberedheteroaryl, 4-10 membered heterocycloalkyl, halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1), wherein said C₁₋₄alkyl, C₁₋₄ alkenyl, C₆₋₁₀ aryl, and 5-10 membered heteroaryl are eachoptionally substituted with 1, 2, or 3 substituents independentlyselected from halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1);

or R³ and R⁴ together with the carbon atoms to which they are attachedform a C₃₋₁₄ cycloalkyl group or 4-14 membered heterocycloalkyl group,each optionally substituted with 1, 2, or 3 substituents independentlyselected from C₁₋₄ alkyl, halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1);

or R³ and R⁵ together with the carbon atoms to which they are attachedform a C₃₋₁₄ cycloalkyl group or 4-14 membered heterocycloalkyl group,each optionally substituted with 1, 2, or 3 substituents independentlyselected from C₁₋₄ alkyl, halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1);

or R⁴ and R⁶ together with the carbon atoms to which they are attachedform a C₃₋₁₄ cycloalkyl group or 4-14 membered heterocycloalkyl group,each optionally substituted with 1, 2, or 3 substituents independentlyselected from C₁₋₄ alkyl, halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1);

or R⁵ and R⁶ together with the carbon atoms to which they are attachedform a C₃₋₁₄ cycloalkyl group or 4-14 membered heterocycloalkyl group,each optionally substituted with 1, 2, or 3 substituents independentlyselected from C₁₋₄ alkyl, halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1);

or R⁷ and R⁹ together with the carbon atoms to which they are attachedform a C₃₋₁₄ cycloalkyl group or 4-14 membered heterocycloalkyl group,each optionally substituted with 1, 2, or 3 substituents independentlyselected from C₁₋₄ alkyl, halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(a1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1);

or R⁷ and R⁹ together with the carbon atoms to which they are attachedform a C₃₋₁₄ cycloalkyl group or 4-14 membered heterocycloalkyl group,each optionally substituted with 1, 2, or 3 substituents independentlyselected from C₁₋₄ alkyl, halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1);

or R⁸ and R¹⁰ together with the carbon atoms to which they are attachedform a C₃₋₁₄ cycloalkyl group or 4-14 membered heterocycloalkyl group,each optionally substituted with 1, 2, or 3 substituents independentlyselected from C₁₋₄ alkyl, halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1);

or R⁹ and R¹⁰ together with the carbon atoms to which they are attachedform a C₃₋₁₄ cycloalkyl group or 4-14 membered heterocycloalkyl group,each optionally substituted with 1, 2, or 3 substituents independentlyselected from C₁₋₄ alkyl, halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1);

Z is C₆₋₁₀ aryl or 5-10 membered heteroaryl; wherein the 5-10 memberedheteroaryl has at least one ring-forming carbon atom and 1, 2, 3, or 4ring-forming heteroatoms independently selected from N, O, and S,wherein the C₆₋₁₀ aryl and 5-10 membered heteroaryl are each optionallysubstituted with 1, 2, or 3 substituents independently selected fromC₁₋₄ alkyl, halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1);

A is H or C₁₋₄ alkyl;

R^(a1), R^(b1), R^(c1), and R^(d1) are each independently selected fromH, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ haloalkyl, OH, CN, NO₂,and CO₂CH₃; wherein said C₁₋₆ alkyl and C₂₋₆ alkenyl are each optionallysubstituted with OH, CN, NO₂, or CO₂CH; and

n is 0, 1, or 2.

Provided herein is a compound of Formula (I):R²-L-R¹  (I)or a pharmaceutically acceptable salt thereof, wherein:

R¹ is a peptide capable of selectively delivering R²L- across a cellmembrane having an acidic or hypoxic mantle;

R² is selected from the group consisting of:

L is a group selected from:

wherein

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each independently selected fromH, C₁₋₄ alkyl, C₁₋₄ alkenyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, halo,CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1),OC(O)R^(b1), OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1), wherein said C₁₋₄alkyl, C₁₋₄ alkenyl, C₆₋₁₀ aryl, and 5-10 membered heteroaryl are eachoptionally substituted with 1, 2, or 3 substituents independentlyselected from halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(a1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1);

or R³ and R⁴ together with the carbon atom to which they are attachedform an C₃₋₇ cycloalkyl group optionally substituted with 1, 2, or 3substituents independently selected from halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

or R³ and R⁵ together with the carbon atom to which they are attachedform an C₃₋₇ cycloalkyl group optionally substituted with 1, 2, or 3substituents independently selected from halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

or R⁴ and R⁶ together with the carbon atom to which they are attachedform an C₃₋₇ cycloalkyl group optionally substituted with 1, 2, or 3substituents independently selected from halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

or R⁵ and R⁶ together with the carbon atom to which they are attachedform an C₃₋₇ cycloalkyl group optionally substituted with 1, 2, or 3substituents independently selected from halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

or R⁷ and R⁸ together with the carbon atom to which they are attachedform an C₃₋₇ cycloalkyl group optionally substituted with 1, 2, or 3substituents independently selected from halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

or R⁷ and R⁹ together with the carbon atom to which they are attachedform an C₃₋₇ cycloalkyl group optionally substituted with 1, 2, or 3substituents independently selected from halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

or R⁸ and R¹⁰ together with the carbon atom to which they are attachedform an C₃₋₇ cycloalkyl group optionally substituted with 1, 2, or 3substituents independently selected from halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

or R⁹ and R¹⁰ together with the carbon atom to which they are attachedform an C₃₋₇ cycloalkyl group optionally substituted with 1, 2, or 3substituents independently selected from halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

A is H or C₁₋₄ alkyl; and

R^(a1), R^(b1), R^(c1), and R^(d1) are each independently selected fromH, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ haloalkyl, OH, CN, NO₂,and CO₂CH₃; wherein said C₁₋₆ alkyl and C₂₋₆ alkenyl are each optionallysubstituted with OH, CN, NO₂, or CO₂CH.

In some embodiments, the lefthand side of L attaches to R² and therighthand side of L attaches to R¹.

In some embodiments, a sulfur atom of the disulfide moiety of L is partof a cysteine residue of R¹.

As used herein, “peptide” refers to a targeting moiety comprising a10-50 amino acid sequence, made up of naturally-occurring amino acidresidues and optionally one or more non-naturally-occurring amino acids.In some embodiments, the peptide of R¹ is a peptide of 20 to 40, 20 to30 amino acids, or 30 to 40 residues. Peptides suitable for use in thecompounds of the invention are those that can insert across a cellmembrane via a conformational change or a change in secondary structurein response to environmental pH changes. In this way, the peptide cantarget acidic tissue and selectively translocate polar, cell-impermeablemolecules across cell membranes in response to low extracellular pH. Insome embodiments, the peptide is capable of selectively delivering aconjugated moiety (e.g., R²L-) across a cell membrane having an acidicor hypoxic mantle having a pH less than about 6.0. In some embodiments,the peptide is capable of selectively delivering a conjugated moiety(e.g., R²L-) across a cell membrane having an acidic or hypoxic mantlehaving a pH less than about 6.5. In some embodiments, the peptide iscapable of selectively delivering a conjugated moiety (e.g., R²L-)across a cell membrane having an acidic or hypoxic mantle having a pHless than about 5.5. In some embodiments, the peptide is capable ofselectively delivering a conjugated moiety (e.g., R²L-) across a cellmembrane having an acidic or hypoxic mantle having a pH between about5.0 and about 6.0.

In certain embodiments, the peptide of R¹ includes a cysteine residuewhich can form the site of attachment to a payload moiety (e.g., R²L-)to be delivered across a cell membrane. In some embodiments, R¹ isattached to L through a cysteine residue of R¹. In some embodiments, thesulfur atom of the cysteine residue can form part of the disulfide bondof the disulfide bond-containing linker L.

Suitable peptides, that can conformationally change based on pH andinsert across a cell membrane, are described, for example, in U.S. Pat.Nos. 8,076,451 and 9,289,508 (each of which is incorporated herein byreference in its entirety). Other suitable peptides are described, forexample, in Weerakkody, et al., PNAS 110 (15), 5834-5839 (Apr. 9, 2013),which is also incorporated herein by reference in its entirety.

In some embodiments, R¹ is a peptide comprising at least one of thefollowing sequences:

(SEQ ID NO. 1; Pv1) ADDQNPWRAYLDLLFPTDTLLLDLLWCG, (SEQ ID NO. 2; Pv2)AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG, and (SEQ ID NO. 3; Pv3)ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG; (SEQ ID NO. 4; Pv4)Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG; (SEQ ID No. 5; Pv5)AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC; and (SEQ ID No. 6; Pv6)AAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG;wherein R¹ is attached to L through a cysteine residue of R¹.

In some embodiments, R¹ is a peptide comprising at least one of thefollowing sequences:

(SEQ ID NO. 1; Pv1) ADDQNPWRAYLDLLFPTDTLLLDLLWCG, (SEQ ID NO. 2; Pv2)AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG, and (SEQ ID NO. 3; Pv3) ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG; and (SEQ ID No. 6; Pv6)AAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG;wherein R¹ is attached to L through a cysteine residue of R¹.

In some embodiments, R¹ is a peptide comprising the sequence

(SEQ ID NO. 1; Pv1) ADDQNPWRAYLDLLFPTDTLLLDLLWCG.

In some embodiments, R¹ is a peptide comprising the sequence

(SEQ ID NO. 2; Pv2) AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG.

In some embodiments, R¹ is a peptide comprising the sequence

(SEQ ID NO. 3; Pv3) ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG.In some embodiments, R¹ is a peptide comprising the sequence

(SEQ ID NO. 4; Pv4) Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG.

In some embodiments, R¹ is a peptide comprising the sequence

(SEQ ID NO. 5; Pv5) AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC.

In some embodiments, R¹ is a peptide comprising the sequence

(SEQ ID NO. 6; Pv6) AAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG.

In some embodiments, R¹ is a peptide consisting of the sequence

(SEQ ID NO. 1; Pv1) ADDQNPWRAYLDLLFPTDTLLLDLLWCG.

In some embodiments, R¹ is a peptide consisting of the sequence

(SEQ ID NO. 2; Pv2) AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG.

In some embodiments, R is a peptide consisting of the sequence

(SEQ ID NO. 3; Pv3) ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG.

In some embodiments, R¹ is a peptide consisting of the sequence Ac-

(SEQ ID NO. 4; Pv4) AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG.

In some embodiments, R¹ is a peptide consisting of the sequence

(SEQ ID NO. 5; Pv5) AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC.

In some embodiments, R¹ is a peptide consisting of the sequence

(SEQ ID NO. 6; Pv6) AAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG.

In some embodiments, R¹ is a peptide comprising at least one sequenceselected from SEQ ID NO: 7 to SEQ ID NO: 311 as shown in Table 1.

In some embodiments, R¹ is a peptide consisting of a sequence selectedfrom SEQ ID NO: 7 to SEQ ID NO: 311 as shown in Table 1.

TABLE 1 Additional R¹ Sequences SEQ ID NO. Sequence 7AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 8GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 9AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 10AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 11GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 12ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG 13ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 14AKEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 15AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG 16AKEQNPIYWARYADWLFTTPLLLLDLALLVDADECT 17ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGTG 18ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG 19GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT 20AAEQNPIYWARYADWLFTTPLLLLALALLVDADEGT 21AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGT 22AAEQNPIYWARYADWLFTTALLLLDLALLVDADEGT 23AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGT 24AAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGT 25AAEQNPIIYWARYADWLFTDLPLLLLDLLALLVDADEGT 26GEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG 27GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEGTCG 28GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG 29GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG 30GGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEGTCG 31AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT 32DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT 33DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADEGCT 34DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 35DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 36AEQNPIYWARYADFLFTTPLLLLDLALLVDADET 37AEQNPIYFARYADWLFTTPLLLLDLALLVDADEGT 38AEQNPIYFARYADFLFTTPLLLLDLALLWDADET 39 AKEDQNPYWARYADWLFTTPLLLLDLALLVDG40 ACEDQNPYWARYADWLFTTPLLLLDLALLVDG 41 AEDQNPYWARYADWLFTTPLLLLDLALLVDCG42 AEDQNPYWARYADWLFTTPLLLLELALLVECG 43 AKEDQNPYWRAYADLFTPLTLLDLLALWDG 44ACEDQNPYWRAYADLFTPLTLLDLLALWDG 45 ACDDQNPWRAYLDLLFPTDTLLLDLLW 46TEDADVLLALDLLLLPTTFLWD 47 AEQNPIYWARYADWLFTTPL 48 AEQNPIYWARYADWLFTTPCL49 ACEQNPIYWARYADWLFTTPL 50 AEQNPIYFARYADWLFTTPL 51KEDQNPWARYADLLFPTTLAW 52 ACEDQNPWARYADLLFPTTLAW 53ACEDQNPWARYADWLFPTTLLLLD 54 ACEEQNPWARYAELLFPTTLAW 55ACEEQNPWARYAEWLFPTTLLLLE 56 ACEEQNPWARYLEWLFPTETLLLEL 57GGEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT 58ACEQNPIY WARYADWLFTTPLLLLDLALLV 59 WARYADWLFTTPLLLLDLALLV DADEGTCG 60WARYADWLFTTPLLLLDLALLV DADEGCT 61GGEQNPIY WARYADWLFTTPLLLLDLALLV DADEGTCG 62ACEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT 63AKEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT 64AKEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT 65AAEQNPIY WARYADWLFTTALLLLDLALLV DADEGT 66ACAEQNPIY WARYADWLFTTGLLLLDLALLV DADEGT 67AEQNPIY WARYADFLFTTALLLLDLALLV DADE_T 68AEQNPIY FARYADWLFTTPLLLLDLALLV DADEGT 69AEQNPIY FARYADFLFTTPLLLLDLALLW DADE_T 70AKEDQNP_Y WARYADWLFTTPLLLLDLALLV DG____ 71ACEDQNP_Y WARYADWLFTTPLLLLDLALLV DG____ 72AEDQNP_Y WARYADWLFTTPLLLLDLALLV DG____ 73AEDQNP_Y WARYADWLFTTPLLLLELALLV ECG___ 74AKEDQNP_Y WRAYAD_LFT PLTLLDLLALW DG____ 75ACEDQNP_Y WRAYAD_LFT PLTLLDLLALW DG____ 76AKEDQNDP_Y WARYADWLFTTPLLLLDLALLV G_____ 77TEDADVLLALDLLLLPTTFLWDAYRAWYPNQECA 78GGEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT 79 AEQNPIY WARYADWLFTTPL 80AEQNPIY WARYADWLFTTPCL 81 ACEQNPIY WARYADWLFTTPL 82ACEQNPIY FARYADWLFTTPL 83 ACDDQNP WRAYLDLLFPTDTLLLDLLW 84ACEEQNP WRAYLELLFPTETLLLELLW 85 ACDDQNP WARYLDWLFPTDTLLLDL 86CDNNNP WRAYLDLLFPTDTLLLDW 87 ACEEQNP WARYLEWLFPTETLLLEL 88ACEDQNP WARYADWLFPTTLLLLD 89 ACEEQNP WARYAEWLFPTTLLLLE 90ACEDQNP WARYADLLFPTTLAW 91 ACEDQNP WARYAELLFPTTLW 92KEDQNP WARYADLLFPTTLW 93 DDDEDNP IYWARYAHWLFTTPLLLLHGALLVDADECT 94DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT 95DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 96DDDEDNPIYWARYAFIWLFTTPLLLLHGALLVNANECT 97DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT 98ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGIG 99ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET 100ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 101GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEGTCG 102GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG 103GGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEGTCG 104AAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGTCG 105AAEQNPIYWARYAEWLFTTPLLLLELALLVDADEGTCG 106GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG 107GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 108GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 109AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 110ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGTG 111DDDEDNPIYWARYAFIWLFTTPLLLLHGALLVNANECT 112DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 113DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT 114DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT 115DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT 116GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT 117AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTCG 118AAEQNPIYWARYAEWLFTTPLLLLELALLVDADEGTCG 119AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG 120GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG 121GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 122GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 123GGEQNPIYWARYADWLFTTPLLLLDALLVNANQGT 124DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 125DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 126ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG 127GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 128GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 129GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG 130AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 131AAEQNPIYWARYADWLFTDLPLLLLDLLALLVDADEGT 132GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG 133GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEGTCG 134AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT 135AEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 136GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 137GGEQNPIYWAQDYAWLFTTPLLLLDLALLDADEGTCG 138GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG 139AAEQNPIYWARYADWLFTTPLLLLALALLVDADEGTCG 140AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG. . . EGTK(rhodamine)C(phalloidin)G 141AAEQNPIYWARYADWLFTTPLLLLELALLDADEGTKCG 142AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 143AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (phalloidin)G 144GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 145ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET 146ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG 147ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 148GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT 149DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 150DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 151GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 152AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (phalloidin)G 153AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG 154AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG 155DDDEDNPIYWARYAHWLFTTPLLLLBGALLVDADECT 156DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT 157DDDEDNPIYWARYAHWLFTTPLLLLBGALLVNADECT 158DDDEDNPIYWARYAHWLFTTPLLLLBGALLVNANECT 159DDDEDNPIYWARYADWLFTTPLLLLIBGALLVDADECT 160DDDEDNPIYWARYADWTFTTPLLLLHGALLVDADECT 161DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT 162DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT 163DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 164DDDEDNPIYWARYHWLFTTPLLLLHGALLVNANECT 165DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 166DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 167DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT 168DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT 169DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT 170GGEQNPIYWARYADWLFTTPLLLLDLALLVNANQGT 171DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 172DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT 173DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT 174DDDEDNPIYWARYAHMLFTTPLLLLDGALLVDADECT 175DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 176DDDEDNPIYWARYAHWLFTTPLLLLDGALLVDADECT 177DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADECT 178DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADECT 179DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 180DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANECT 181AAEQNPIYWARYADWLFTTGLLLLDLALLVDADEGT 182GGEQNPIYWARYAWDLFTTPLLLLDLALLVDADEGTCG 183GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG 184GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 185GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 186AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 187GGEQNPIYWARYADWLFTTPLLLLDALLVDADEGTCG 188GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEGTCG 189GGEQNPIYWARYADWLFTTPLLLDLLALLVDADEGTCG 190GGEQNPIYWARYADWLFTTPLLLLLDALLVDADEGTCG 191GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG 192GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 193GGEQNP1YWAQDYAWLFTTPLLLLDLALLVDADEGTCG 194GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 195GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 196GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG 197AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 198GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 199GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 200GGEQNPIYWAQYADWLFTTPLLLLDLALLVDADEGTCG 201AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 202AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG 203. . . EGTK(rhidamine)C(phalloidin)G 204AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG 205ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG 206AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC (phalloidin)G 207AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG 208AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG 209AAEQNPIYWARYADWLFTDLPLLLLDLLALLVDADEGT 210AAEQNPIYWARYAAWLFTTPLLLLDLALLVDADEGTCG 211GGEQNPIYWAQYDAWLFTTPLLLLDLALLVDADEGTCG 212GGEQNPIYWAQDYAWLFTTPLLLLDLALLVDADEGTCG 213GGEQNPIYWARYDAWLFTTPLLLLDLALLVDADEGTCG 214AAEQNPIYWARYAEWLFTTPLLLLDLALLVDADEGTCG 215AAEQNPIYWARYAEWLFTTPLLLLELALLVDADEGTCG 216AAEQNPIYWARYADWLFTTPLLLLALALLVDADEGTCG 217AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTCG 218AAEQNPIYWARYAEWLFTTPLLLLELALLVDADEGTCG 219AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG 220ACEQNPIYWARYAKWLFTTPLLLLKLALLVDADEGTG 221ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGTG 222AAEQNPIYWARYADWLFTTALLLLDLALLVDADEGT 223 AEQNPIYFARYADLLFPTTLAW 224AEQNPIYWARYADLLFPTTLAF 225 AEQNPIYWARYADLLFPTTLAW 226ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET 227GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT 228AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 229AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG 230AKEQNPIYWARYADWLFTTPLLLLDLALLVDADECT 231 CCTCTTACCTCAGTTACA 232D-Arg8D-Arg8-CCTCTTACCTCAGTTACA 233 D-Lys4D-Lys4-CCTCTTACCTCAGTTACA 234S-S-CCTCTTACCTCAGTTACA 235 S-S-CCTCTGACCTCATTTACA 236D-Arg8-DecaD-Arg8-Deca-CCTCTTACCTCAGTTACA 237D-Arg8-Deca-mismatchD-Arg8-Deca- CCTCTGACCTCATTTACA 238S-S-CCTCTTACCTCAGTTACA 239 AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 240AEDQNPYWARYDWLFTTPLLLLDLALLVDCG 241 AEDQNPYWARYADWLFTTPLLLLELALLVECG 242AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGCT 243ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET 244AE-QN-PIYWARYADWLFTTPLLLLDLALLVDADEGT-COOH 245AEDQN-P-YWARYADWLFTTPLLLLDLALLVD---G--COOH 246AEDQNDP-YWARYADWLFTTPLLLLDLALLV----G--COOH 247AEQNPIYWARYADFLFTTPLLLLDLALLV DADET-COOH 248AEQNPI YFARYADWLFTTPLLLLDLALLV DADET-COOH 249AEQNPI YFARYADFLFTTPLLLLDLALLW DADET-COOH 250AE-QN-PI YWARYADWLFTTPLLLLDLALLV DADEGCT- COOH 251AEDQN-PI YWARYADWLFTTPLLLLDLALLV DC--G-T- COOH 252AEDQNDPI YWARYADWLFTTPLLLLELALLV EC--G-T- COOH 253Chelate-ACEEQNPWARYLEWLFPTETLLLEL 254AEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT-COOH 255AKEDQNPY WARYADWLFTTPLLLLDLALLV DG-COOH 256AKEDQNDPY WARYADWLFTTPLLLLDLALLV G-COOH 257AEQNPI YWARYADWLFTTPLLLLDLALLV DADEGC- Biotin-T-COO H 258AEDQNP YWARYADWLFTTPLLLLDLALLV DC-Biotin- G-COOH 259AEDQNP YWARYADWLFTTPLLLLELALLV EC-Biotin- G-COOH 260ACEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT 261ACEDQNPY WARYADWLFTTPLLLLDLALLV DG 262 ACEDQNPY WRAYADLFTPLTLLDLLALW DG263 ACDDQNP WRAYLDLLFPTDTLLLDLLW 264 WRAYLELLFPTETLLLELLW 265WARYLDWLFPTDTLLLDL 266 WRAYLDLLFPTDTLLLDW 267 WARYLEWLFPTETLLLEL 268WAQYLELLFPTETLLLEW 269 WRAYLELLFPTETLLLEW 270 WARYADWLFPTTLLLLD 271WARYAEWLFPTTLLLLE 272 ACEDQNP WARYADLLFPTTLAW 273ACEEQNP WARYAELLFPTTLAW 274 Ac-TEDADVLLALDLLLLPTTFLWDAYRAWYPNQECA-Am 275CDDDDDNPNY WARYANWLFTTPLLLLNGALLV EAEET 276CDDDDDNPNY WARYAPWLFTTPLLLLPGALLV EAEET 277Ac-AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGCT 278Ac-AKEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTG 279ACEQNPIYWARYANWLFTTPLLLLNLALLVDADEGT 280Ac-AAEQNPIYWARYADWLFTTPLLLLELALLVDADEGTKCG 281DDDEDNPIYWARYADWLFTTPLLLLHGALLVDADET 282CDDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADET 283DDDEDNPIYWARYAHWLFTTPLLLLHGALLVDADEGT 284DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNADECT 285DDDEDNPIYWARYAHWLFTTPLLLLHGALLVNANEGT 286AKEDQNDPYWARYADWLFTTPLLLLDLALLVG 287 AEDQNPYWARYADWLFTTPLLLLELALLVCG 288AKDDQNPWRAYLDLLFPTDTLLLDLLWC 289 ACEEQNPWRAYLELLFPTETLLLELLW 290ACDDQNPWARYLDWLFPTDTLLLDL 291 CDNNNPWRAYLDLLFPTDTLLLDW 292CEEQQPWAQYLELLFPTETLLLEW 293 EEQQPWRAYLELLFPTETLLLEW 294CDDDDDNPNYWARYANWLFTTPLLLLNGALLVEAEET 295CDDDDDNPNYWARYAPWLFTTPLLLLPGALLVEAEE 296 AEQNPIYFARYADLLFPTTLAW 297AEQNPIYWARYADLLFPTTLAF 298 AEQNPIYWARYADLLFPTTLAW 299KEDQNPWARYADLLFPTTLW 300 ACEEQNPQAEYAEWLFPTTLLLLE 301AAEEQNPWARYLEWLFPTETLLLEL 302 AKEEQNPWARYLEWLFPTETLLLEL 303AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTGG 304XXEXNPIYWAXXXXXLFTXXLLLXXXALLVXAXXXTXG 305DAAEQNPIYWARYADWLFTTLPLLLLDLLALLVDADEGTKGG 306GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTGG 307XXEXNPIYWAXXXXXLFTXXLLLXXXALLVXAXXXTGG 308DGGEQNDPIYWARYADWLFTTLPLLLLDLLALLVDA DEGCTXGG 309AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTCG 310AEDQNPYWARYDWLFTTPLLLLDLALLVDCG 311 GLAGLAGLLGLEGLLGLPLGLLEGLWLGLELEGN

Any of the recited peptides useful in the present invention can bemodified to include a cysteine residue by replacing a non-cysteineresidue with cysteine, or appending a cysteine residue to either theN-terminus or C-terminus.

In some embodiments, the peptide of R¹ is a conformationally restrictedpeptide. A conformationally restricted peptide can include, for example,macrocyclic peptides and stapled peptides. A stapled peptide is apeptide constrained by a covalent linkage between two amino acidside-chains, forming a peptide macrocycle. Conformationally restrictedpeptides are described, for example, in Guerlavais et al., AnnualReports in Medicinal Chemistry 2014, 49, 331-345; Chang et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica (2013), 110(36), E3445-E3454; Tesauro et al., Molecules 2019,24, 351-377; Dougherty et al., Journal of Medicinal Chemistry (2019),62(22), 10098-10107; and Dougherty et al., Chemical Reviews (2019),119(17), 10241-10287, each of which is incorporated herein by referencein its entirety.

In some embodiments, R¹ is a peptide having 10 to 50 amino acids. Insome embodiments, R¹ is a peptide having 20 to 40 amino acids. In someembodiments, R¹ is a peptide having 20 to 40 amino acids. In someembodiments, R¹ is a peptide having 10 to 20 amino acids. In someembodiments, R¹ is a peptide having 20 to 30 amino acids. In someembodiments, R¹ is a peptide having 30 to 40 amino acids.

Suitable small molecule microtubule targeting moieties (e.g., R²) can becytotoxic compounds like maytansinoids that may have undesirable sideeffects when delivered systemically because of their possibledeleterious effect on normal tissue. Small molecule microtubuletargeting agents include, but are not limited to, maytansinoids,aclitaxel, docetaxel, epothilones, discodermolide, the vinca alkaloids,colchicine, combretastatins, and derivatives and analogues of theaforementioned. Microtubule targeting agents are described in Tangutur,A. D., Current Topics in Medicinal Chemistry, 2017 17(22): 2523-2537.Microtubule-targeting agents also include maytansinoids, such asmaytansine (DM1) and derivatives and analogues thereof, which aredescribed in Lopus, M, Cancer Lett., 2011, 307(2): 113-118; andWiddison, W., J. Med. Chem. 2006, 49:4392-4408.

In some embodiments, R² is the following group:

In some embodiments, R² is the following group:

In some embodiments, R² is the following group:

In some embodiments, R² is the following group:

In some embodiments, R² is the following group:

In some embodiments, R² is a maytansinoid. In some embodiments, R² isDM1 or DM4. In some embodiments, R² is DM1. In some embodiments, R² isDM4.

In some embodiments, L is a linking moiety that covalently connects R¹and R², and functions to release a moiety containing R² in the vicinityof acidic or hypoxic tissue, such as inside a cell of diseased tissue.

In some embodiments, L is a linking chain of 1 to 40, 1 to 30, 1 to 25,1 to 20, 1 to 15, 1 to 10, or 1 to 5 chain atoms (including both carbonand heteroatoms), which is optionally substituted with 1-10 R^(q)substituents, and wherein one or more chain carbon atoms of L can beoxidized to form a carbonyl (C═O), and wherein one or more N and S chainatoms can each be optionally oxidized to form an amine oxide, sulfoxideor sulfonyl group; wherein

each R^(q) is independently selected from OH, CN, —COOH, NH₂, halo, C₁₋₆haloalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy, C₁₋₆ alkylthio,phenyl, 5-6 membered heteroaryl, 4-6 membered heterocycloalkyl, C₃₋₆cycloalkyl, NH(C₁₋₆ alkyl) and N(C₁₋₆ alkyl)₂, wherein the C₁₋₆ alkyl,phenyl, C₃₋₆ cycloalkyl, 4-6 membered heterocycloalkyl, and 5-6 memberedheteroaryl of R^(q) are each optionally substituted with halo, OH, CN,—COOH, NH₂, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₄ haloalkyl, C₁₋₄ haloalkoxy,phenyl, C₃₋₁₀ cycloalkyl, 5- or 6-membered heteroaryl or 4-6 memberedheterocycloalkyl; and

two R^(q) groups together with the chain atoms to which they areattached can form a phenyl, 5-6 membered heteroaryl, 4-6 memberedheterocycloalkyl, or C₃₋₆ cycloalkyl ring.

In some embodiments, R^(q) is independently selected from OH, CN, —COOH,NH₂, halo, C₁₋₆ haloalkyl, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkoxy,NH(C₁₋₆ alkyl) and N(C₁₋₆ alkyl)₂.

In some embodiments, L is the following group:

In some embodiments, L is the following group:

In some embodiments, L is the following group:

In some embodiments, n is 0. In some embodiments, n is 1. In someembodiments, n is 2.

In some embodiments, L is the following group:

In some embodiments, L is the following group:

In some embodiments, L is the following group:

In some embodiments, L is the following group:

In some embodiments, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are eachindependently selected from H and C₁₋₄ alkyl. In some embodiments, R³,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each H.

In some embodiments, R³ and R⁴ are each independently selected from Hand C₁₋₄ alkyl.

In some embodiments, R³ and R⁴ are each H.

In some embodiments, R⁵ and R⁶ are each independently selected from Hand C₁₋₄ alkyl.

In some embodiments, R⁵ and R⁶ are each H.

In some embodiments, R⁷ and R⁸ are each independently selected from Hand C₁₋₄ alkyl.

In some embodiments, R⁷ and R⁸ are each H.

In some embodiments, R⁹ and R¹⁰ are each independently selected from Hand C₁₋₄ alkyl.

In some embodiments, R⁹ and R¹⁰ are each H.

In some embodiments, A is H. In some embodiments, A is C₁₋₄ alkyl. Insome embodiments, A is CH₃.

In some embodiments, Z is C₆₋₁₀ aryl, optionally substituted with 1, 2,or 3 substituents independently selected from C₁₋₄ alkyl, halo, CN, NO₂,OR^(a1), SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1),OC(O)R^(b1), OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1).

In some embodiments, Z is phenyl, optionally substituted with 1, 2, or 3substituents independently selected from C₁₋₄ alkyl, halo, CN, NO₂,OR^(a1), SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1),OC(O)R^(b1), OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1).

In some embodiments, Z is phenyl.

In some embodiments, the compound of the invention is a compound ofFormula (II):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is a peptide;

R² is a a small molecule microtubule targeting moiety;

A is H or C₁₋₄ alkyl;

Ring Y is a monocyclic C₅₋₇ cycloalkyl ring or a monocyclic 5-7 memberedheterocycloalkyl ring;

each R is independently selected from C₁₋₄ alkyl, halo, CN, NO₂,OR^(a1), SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1),OC(O)R^(b1), OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

or two adjacent R together with the atoms to which they are attachedform a fused monocyclic C₅₋₇ cycloalkyl ring, a fused monocyclic 5-7membered heterocycloalkyl ring, a fused C₆₋₁₀ aryl ring, or a fused 6-10membered heteroaryl ring, each of which is optionally substituted with1, 2, or 3 substituents independently selected from C₁₋₄ alkyl, halo,CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1),OC(O)R^(b1), OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1);

R^(a1), R^(b1), R^(c1), and R^(d1) are each independently selected fromH, C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, each optionally substitutedwith 1, 2, or 3 substituents independently selected from halo, OH, CN,and NO₂; and

m is 0, 1, 2, or 3.

In some embodiments of compounds of Formula (II), R¹ is a peptidecomprising the sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, or SEQ ID NO:5.

In some embodiments of compounds of Formula (II), R¹ is Pv1, Pv2, Pv3,Pv4, or Pv5.

In some embodiments of compounds of Formula (II), R¹ is attached to thecore via a cysteine residue of R¹ wherein one of the sulfur atoms of thedisulfide moiety in Formula II is derived from the cysteine residue.

In some embodiments of compounds of Formula (II), R² is a maytansinoid.In some embodiments of Formula (II), R² is DM1 or DM4. In someembodiments of Formula (II), R² is DM1. In some embodiments of Formula(II), R² is DM4.

In some embodiments of compounds of Formula (II), R² is the followinggroup:

In some embodiments of compounds of Formula (II), R² is the followinggroup:

In some embodiments of compounds of Formula (II), R² is the followinggroup:

In some embodiments of compounds of Formula (II), R² is the followinggroup:

In some embodiments of compounds of Formula (II), A is H. In someembodiments of compounds of Formula (II), A is C₁₋₄ alkyl. In someembodiments of compounds of Formula (II), A is CH₃.

In some embodiments of compounds of Formula (II), Ring Y is a monocyclicC₅₋₇ cycloalkyl ring.

In some embodiments of compounds of Formula (II), Ring Y is acyclopentyl ring.

In some embodiments of compounds of Formula (II), Ring Y is a cyclohexylring.

In some embodiments of compounds of Formula (II), Ring Y is acycloheptyl ring.

In some embodiments of compounds of Formula (II), Ring Y is a monocyclic5-7 membered heterocycloalkyl ring.

In some embodiments of compounds of Formula (II), Ring Y is a 5-memberedheterocycloalkyl ring.

In some embodiments of compounds of Formula (II), Ring Y is a 6-memberedheterocycloalkyl ring.

In some embodiments of compounds of Formula (II), Ring Y is a 7-memberedheterocycloalkyl ring.

In some embodiments of compounds of Formula (II), two adjacent Rtogether with the atoms to which they are attached form a fusedmonocyclic C₅₋₇ cycloalkyl ring, a fused monocyclic 5-7 memberedheterocycloalkyl ring, a fused C₆₋₁₀ aryl ring, or a fused 6-10 memberedheteroaryl ring, each of which is optionally substituted with 1, 2, or 3substituents independently selected from C₁₋₄ alkyl, halo, CN, NO₂,OR^(a1), SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1),OC(O)R^(b1), OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1).

In some embodiments of compounds of Formula (II), m is 0.

In some embodiments of compounds of Formula (II), m is 1.

In some embodiments of compounds of Formula (II), m is 2.

In some embodiments of compounds of Formula (II), m is 3.

In some embodiments, the compounds of the invention is a compound ofFormula (III), Formula (IV), or Formula (V):

or a pharmaceutically acceptable salt thereof, wherein R¹, R², R, A, andm are defined as in any of the embodiments above for Formula (II).

In some embodiments, the compound of formula (I) is selected from:

or a pharmaceutically acceptable salt of any of the aforementioned.

In some embodiments, the compound of Formula (I) is selected from:

or a pharmaceutically acceptable salt of any of the aforementioned.

In some embodiments, provided herein is a compound having Formula (I-A):

or a salt thereof, wherein Cy¹ is C₆₋₁₀ aryl or 5-10 memberedheteroaryl. In some embodiments, Cy¹ is pyridyl.

In some embodiments, provided herein is a compound having Formula (I-B):

or a salt thereof, wherein Cy¹ is C₆₋₁₀ aryl or 5-10 memberedheteroaryl. In some embodiments, Cy¹ is pyridyl.

The molecules of the invention can be tagged, for example, with a probesuch as a fluorophore, radioisotope, and the like. In some embodiments,the probe is a fluorescent probe, such as LICOR. A fluorescent probe caninclude any moiety that can re-emit light upon light excitation (e.g., afluorophore).

The Amino acids are represented by the IUPAC abbreviations, as follows:Alanine (Ala; A), Arginine (Arg; R), Asparagine (Asn; N), Aspartic acid(Asp; D), Cysteine (Cys; C), Glutamine (Gln; Q), Glutamic acid (Glu; E),Glycine (Gly; G), Histidine (His; H), Isoleucine (Ile; I), Leucine (Leu;L), Lysine (Lys; K), Methionine (Met; M), Phenylalanine (Phe; F),Proline (Pro; P), Serine (Ser; S), Threonine (Thr; T), Tryptophan (Trp;W), Tyrosine (Tyr; Y), Valine (Val; V).

The term “Pv1” means ADDQNPWRAYLDLLFPTDTLLLDLLWCG, which is the thepeptide of SEQ ID No. 1.

The term “Pv2” means AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG, which is thepeptide of SEQ ID No. 2.

The term “Pv3” means ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG, which is thepeptide of SEQ ID No. 3.

The term “Pv4” means Ac-AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTKCG, whichis the peptide of SEQ ID NO. 4.

The term “Pv5” means AAEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTC, which is thepeptide of SEQ ID NO. 5. The term “Pv6” meansAAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG, which is the peptide of SEQ IDNO. 6. In the compounds of the invention, the peptides R¹ are attachedto the disulfide linker by a cysteine moiety.

The term “acidic and/or hypoxic mantle” refers to the environment of thecell in the diseased tissue in question having a pH lower than 7.0 andpreferably lower than 6.5. An acidic or hypoxic mantle more preferablyhas a pH of about 5.5 and most preferably has a pH of about 5.0. Thecompounds of formula (I) insert across a cell membrane having an acidicand/or hypoxic mantle in a pH dependent fashion to insert R²L into thecell, whereupon the disulfide bond of the linker is cleaved to deliverfree R²L (or R²L*, wherein L* is a product of degradation). Since thecompounds of formula (I) are pH-dependent, they preferentially insertacross a cell membrane only in the presence of an acidic or hypoxicmantle surrounding the cell and not across the cell membrane of “normal”cells, which do not have an acidic or hypoxic mantle.

The terms “pH-sensitive” or “pH-dependent” as used herein to refer tothe peptide R¹ or to the mode of insertion of the peptide R¹ or of thecompounds of the invention across a cell membrane, means that thepeptide has a higher affinity to a cell membrane lipid bilayer having anacidic or hypoxic mantle than a membrane lipid bilayer at neutral pH.Thus, the compounds of the invention preferentially insert through thecell membrane to insert R²L to the interior of the cell (and thusdeliver R²H as described above) when the cell membrane lipid bilayer hasan acidic or hypoxic mantle (a “diseased” cell) but does not insertthrough a cell membrane when the mantle (the environment of the cellmembrane lipid bilayer) is not acidic or hypoxic (a “normal” cell). Itis believed that this preferential insertion is achieved as a result ofthe peptide R¹ forming a helical configuration, which facilitatesmembrane insertion.

The term “small molecule microtubule targeting moiety” refers to achemical group that binds to microtubules. The small moleculemicrotubule targeting moiety can be a group derived from a compound thatinhibits the activity of microtubules. For example, the small moleculemicrotubule targeting moiety may suppress the dynamic stability ofmicrotubules. In some embodiments, the small molecule microtubuletargeting moiety has a molecular weight (Da) of about 100-1500, about100-800, about 500-1,000, about 600-1,000, about 100-500, about 700-900,or about 250-500.

It is further appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, canalso be provided in combination in a single embodiment (while theembodiments are intended to be combined as if written in multiplydependent form). Conversely, various features of the invention whichare, for brevity, described in the context of a single embodiment, canalso be provided separately or in any suitable subcombination. Thus, itis contemplated as features described as embodiments of the compounds ofFormula (I) can be combined in any suitable combination.

At various places in the present specification, certain features of thecompounds are disclosed in groups or in ranges. It is specificallyintended that such a disclosure include each and every individualsubcombination of the members of such groups and ranges. For example,the term “C₁₋₆ alkyl” is specifically intended to individually disclose(without limitation) methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl and C₆alkyl.

The term “n-membered,” where n is an integer, typically describes thenumber of ring-forming atoms in a moiety where the number ofring-forming atoms is n. For example, piperidinyl is an example of a6-membered heterocycloalkyl ring, pyrazolyl is an example of a5-membered heteroaryl ring, pyridyl is an example of a 6-memberedheteroaryl ring and 1,2,3,4-tetrahydro-naphthalene is an example of a10-membered cycloalkyl group.

At various places in the present specification, variables definingdivalent linking groups may be described. It is specifically intendedthat each linking substituent include both the forward and backwardforms of the linking substituent. For example, —NR(CR′R″)_(n)— includesboth —NR(CR′R″)_(n)— and —(CR′R″)_(n)NR— and is intended to discloseeach of the forms individually. Where the structure requires a linkinggroup, the Markush variables listed for that group are understood to belinking groups. For example, if the structure requires a linking groupand the Markush group definition for that variable lists “alkyl” or“aryl” then it is understood that the “alkyl” or “aryl” represents alinking alkylene group or arylene group, respectively.

The term “substituted” means that an atom or group of atoms formallyreplaces hydrogen as a “substituent” attached to another group. The term“substituted”, unless otherwise indicated, refers to any level ofsubstitution, e.g., mono-, di-, tri-, tetra- or penta-substitution,where such substitution is permitted. The substituents are independentlyselected, and substitution may be at any chemically accessible position.It is to be understood that substitution at a given atom is limited byvalency. It is to be understood that substitution at a given atomresults in a chemically stable molecule. The phrase “optionallysubstituted” means unsubstituted or substituted. The term “substituted”means that a hydrogen atom is removed and replaced by a substituent. Asingle divalent substituent, e.g., oxo, can replace two hydrogen atoms.

The term “C_(n-m)” indicates a range which includes the endpoints,wherein n and m are integers and indicate the number of carbons.Examples include C₁₋₄, C₁₋₆ and the like.

The term “alkyl” employed alone or in combination with other terms,refers to a saturated hydrocarbon group that may be straight-chained orbranched. The term “C_(n-m) alkyl”, refers to an alkyl group having n tom carbon atoms. An alkyl group formally corresponds to an alkane withone C—H bond replaced by the point of attachment of the alkyl group tothe remainder of the compound. In some embodiments, the alkyl groupcontains from 1 to 6 carbon atoms, from 1 to 4 carbon atoms, from 1 to 3carbon atoms, or 1 to 2 carbon atoms. Examples of alkyl moietiesinclude, but are not limited to, chemical groups such as methyl, ethyl,n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higherhomologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl,1,2,2-trimethylpropyl and the like.

The term “alkenyl” employed alone or in combination with other terms,refers to a straight-chain or branched hydrocarbon group correspondingto an alkyl group having one or more double carbon-carbon bonds. Analkenyl group formally corresponds to an alkene with one C—H bondreplaced by the point of attachment of the alkenyl group to theremainder of the compound. The term “C_(n-m) alkenyl” refers to analkenyl group having n to m carbons. In some embodiments, the alkenylmoiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms. Example alkenylgroups include, but are not limited to, ethenyl, n-propenyl,isopropenyl, n-butenyl, sec-butenyl and the like.

The term “alkynyl” employed alone or in combination with other terms,refers to a straight-chain or branched hydrocarbon group correspondingto an alkyl group having one or more triple carbon-carbon bonds. Analkynyl group formally corresponds to an alkyne with one C—H bondreplaced by the point of attachment of the alkyl group to the remainderof the compound. The term “C_(n-m) alkynyl” refers to an alkynyl grouphaving n to m carbons. Example alkynyl groups include, but are notlimited to, ethynyl, propyn-1-yl, propyn-2-yl and the like. In someembodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3carbon atoms.

The term “alkylene”, employed alone or in combination with other terms,refers to a divalent alkyl linking group. An alkylene group formallycorresponds to an alkane with two C—H bond replaced by points ofattachment of the alkylene group to the remainder of the compound. Theterm “C_(n-m) alkylene” refers to an alkylene group having n to m carbonatoms. Examples of alkylene groups include, but are not limited to,ethan-1,2-diyl, ethan-1,1-diyl, propan-1,3-diyl, propan-1,2-diyl,propan-1,1-diyl, butan-1,4-diyl, butan-1,3-diyl, butan-1,2-diyl,2-methyl-propan-1,3-diyl and the like.

The term “amino” refers to a group of formula —NH₂.

The term “carbonyl”, employed alone or in combination with other terms,refers to a —C(═O)— group, which also may be written as C(O).

The term “cyano” or “nitrile” refers to a group of formula —C—N, whichalso may be written as —CN.

The terms “halo” or “halogen”, used alone or in combination with otherterms, refers to fluoro, chloro, bromo and iodo. In some embodiments,“halo” refers to a halogen atom selected from F, Cl, or Br. In someembodiments, halo groups are F.

The term “haloalkyl” as used herein refers to an alkyl group in whichone or more of the hydrogen atoms has been replaced by a halogen atom.The term “C_(n-m) haloalkyl” refers to a C_(n-m) alkyl group having n tom carbon atoms and from at least one up to {2(n to m)+1} halogen atoms,which may either be the same or different. In some embodiments, thehalogen atoms are fluoro atoms. In some embodiments, the haloalkyl grouphas 1 to 6 or 1 to 4 carbon atoms. Example haloalkyl groups include CF₃,C₂F₅, CHF₂, CH₂F, CCl₃, CHCl₂, C₂Cl₅ and the like. In some embodiments,the haloalkyl group is a fluoroalkyl group.

The term “haloalkoxy”, employed alone or in combination with otherterms, refers to a group of formula —O-haloalkyl, wherein the haloalkylgroup is as defined above. The term “C_(n-m) haloalkoxy” refers to ahaloalkoxy group, the haloalkyl group of which has n to m carbons.

Example haloalkoxy groups include trifluoromethoxy and the like. In someembodiments, the haloalkoxy group has 1 to 6, 1 to 4, or 1 to 3 carbonatoms.

The term “oxo” refers to an oxygen atom as a divalent substituent,forming a carbonyl group when attached to carbon, or attached to aheteroatom forming a sulfoxide or sulfone group, or an N-oxide group. Insome embodiments, heterocyclic groups may be optionally substituted by 1or 2 oxo (═O) substituents.

The term “oxidized” in reference to a ring-forming N atom refers to aring-forming N-oxide.

The term “oxidized” in reference to a ring-forming S atom refers to aring-forming sulfonyl or ring-forming sulfinyl.

The term “aromatic” refers to a carbocycle or heterocycle having one ormore polyunsaturated rings having aromatic character (i.e., having(4n+2) delocalized Q (pi) electrons where n is an integer).

The term “aryl,” employed alone or in combination with other terms,refers to an aromatic hydrocarbon group, which may be monocyclic orpolycyclic (e.g., having 2 fused rings). The term “C_(n-m) aryl” refersto an aryl group having from n to m ring carbon atoms. Aryl groupsinclude, e.g., phenyl, naphthyl, and the like. In some embodiments, arylgroups have from 6 to about 10 carbon atoms. In some embodiments arylgroups have 6 carbon atoms. In some embodiments aryl groups have 10carbon atoms. In some embodiments, the aryl group is phenyl.

The term “heteroaryl” or “heteroaromatic,” employed alone or incombination with other terms, refers to a monocyclic or polycyclicaromatic heterocycle having at least one heteroatom ring member selectedfrom sulfur, oxygen and nitrogen. In some embodiments, the heteroarylring has 1, 2, 3 or 4 heteroatom ring members independently selectedfrom nitrogen, sulfur and oxygen. In some embodiments, any ring-formingN in a heteroaryl moiety can be an N-oxide. In some embodiments, theheteroaryl has 5-14 ring atoms including carbon atoms and 1, 2, 3 or 4heteroatom ring members independently selected from nitrogen, sulfur andoxygen. In some embodiments, the heteroaryl has 5-10 ring atomsincluding carbon atoms and 1, 2, 3 or 4 heteroatom ring membersindependently selected from nitrogen, sulfur and oxygen. In someembodiments, the heteroaryl has 5-6 ring atoms and 1 or 2 heteroatomring members independently selected from nitrogen, sulfur and oxygen. Insome embodiments, the heteroaryl is a five-membered or six-memberedheteroaryl ring. In other embodiments, the heteroaryl is aneight-membered, nine-membered or ten-membered fused bicyclic heteroarylring.

A five-membered heteroaryl ring is a heteroaryl group having five ringatoms wherein one or more (e.g., 1, 2 or 3) ring atoms are independentlyselected from N, O and S.

A six-membered heteroaryl ring is a heteroaryl group having six ringatoms wherein one or more (e.g., 1, 2 or 3) ring atoms are independentlyselected from N, O and S.

The term “cycloalkyl,” employed alone or in combination with otherterms, refers to a non-aromatic hydrocarbon ring system (monocyclic,bicyclic or polycyclic), including cyclized alkyl and alkenyl groups.The term “C_(n-m) cycloalkyl” refers to a cycloalkyl that has n to mring member carbon atoms. Cycloalkyl groups can include mono- orpolycyclic (e.g., having 2, 3 or 4 fused rings) groups and spirocycles.Cycloalkyl groups can have 3, 4, 5, 6 or 7 ring-forming carbons (C₃₋₇).In some embodiments, the cycloalkyl group has 3 to 6 ring members, 3 to5 ring members, or 3 to 4 ring members. In some embodiments, thecycloalkyl group is monocyclic. In some embodiments, the cycloalkylgroup is monocyclic or bicyclic. In some embodiments, the cycloalkylgroup is a C₃₋₆ monocyclic cycloalkyl group. Ring-forming carbon atomsof a cycloalkyl group can be optionally oxidized to form an oxo orsulfido group. Cycloalkyl groups also include cycloalkylidenes. In someembodiments, cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl. Also included in the definition of cycloalkyl are moietiesthat have one or more aromatic rings fused (i.e., having a bond incommon with) to the cycloalkyl ring, e.g., benzo or thienyl derivativesof cyclopentane, cyclohexane and the like. A cycloalkyl group containinga fused aromatic ring can be attached through any ring-forming atomincluding a ring-forming atom of the fused aromatic ring. Examples ofcycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl,and the like. In some embodiments, the cycloalkyl group is cyclopropyl,cyclobutyl, cyclopentyl, or cyclohexyl.

The term “heterocycloalkyl,” employed alone or in combination with otherterms, refers to a non-aromatic ring or ring system, which mayoptionally contain one or more alkenylene groups as part of the ringstructure, which has at least one heteroatom ring member independentlyselected from nitrogen, sulfur, oxygen and phosphorus, and which has4-10 ring members, 4-7 ring members, or 4-6 ring members. Includedwithin the term “heterocycloalkyl” are monocyclic 4-, 5-, 6- and7-membered heterocycloalkyl groups. Heterocycloalkyl groups can includemono- or bicyclic (e.g., having two fused or bridged rings) orspirocyclic ring systems. In some embodiments, the heterocycloalkylgroup is a monocyclic group having 1, 2 or 3 heteroatoms independentlyselected from nitrogen, sulfur and oxygen. Ring-forming carbon atoms andheteroatoms of a heterocycloalkyl group can be optionally oxidized toform an oxo or sulfido group or other oxidized linkage (e.g., C(O),S(O), C(S) or S(O)₂, N-oxide etc.) or a nitrogen atom can bequaternized. The heterocycloalkyl group can be attached through aring-forming carbon atom or a ring-forming heteroatom. In someembodiments, the heterocycloalkyl group contains 0 to 3 double bonds. Insome embodiments, the heterocycloalkyl group contains 0 to 2 doublebonds. Also included in the definition of heterocycloalkyl are moietiesthat have one or more aromatic rings fused (i.e., having a bond incommon with) to the heterocycloalkyl ring, e.g., benzo or thienylderivatives of piperidine, morpholine, azepine, etc. A heterocycloalkylgroup containing a fused aromatic ring can be attached through anyring-forming atom including a ring-forming atom of the fused aromaticring. Examples of heterocycloalkyl groups include 2-pyrrolidinyl,morpholinyl, azetidinyl, tetrahydrofuranyl, tetrahydropyranyl, andpiperazinyl.

At certain places, the definitions or embodiments refer to specificrings (e.g., an azetidine ring, a pyridine ring, etc.). Unless otherwiseindicated, these rings can be attached to any ring member provided thatthe valency of the atom is not exceeded. For example, an azetidine ringmay be attached at any position of the ring, whereas an azetidin-3-ylring is attached at the 3-position.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent invention that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically inactive startingmaterials are known in the art, such as by resolution of racemicmixtures or by stereoselective synthesis. Many geometric isomers ofolefins, C═N double bonds and the like can also be present in thecompounds described herein, and all such stable isomers are contemplatedin the present invention. Cis and trans geometric isomers of thecompounds of the present invention are described and may be isolated asa mixture of isomers or as separated isomeric forms.

Resolution of racemic mixtures of compounds can be carried out by any ofnumerous methods known in the art. One method includes fractionalrecrystallization using a chiral resolving acid which is an opticallyactive, salt-forming organic acid. Suitable resolving agents forfractional recrystallization methods are, e.g., optically active acids,such as the D and L forms of tartaric acid, diacetyltartaric acid,dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or thevarious optically active camphorsulfonic acids such as α-camphorsulfonicacid. Other resolving agents suitable for fractional crystallizationmethods include stereoisomerically pure forms of α-methylbenzylamine(e.g., S and R forms, or diastereomerically pure forms),2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,cyclohexylethylamine, 1,2-diaminocyclohexane and the like.

Resolution of racemic mixtures can also be carried out by elution on acolumn packed with an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent composition canbe determined by one skilled in the art.

In some embodiments, the compounds of the invention have the(R)-configuration. In other embodiments, the compounds have the(S)-configuration. In compounds with more than one chiral centers, eachof the chiral centers in the compound may be independently (R) or (S),unless otherwise indicated.

Compounds of the invention also include tautomeric forms. Tautomericforms result from the swapping of a single bond with an adjacent doublebond together with the concomitant migration of a proton. Tautomericforms include prototropic tautomers which are isomeric protonationstates having the same empirical formula and total charge. Exampleprototropic tautomers include ketone-enol pairs, amide-imidic acidpairs, lactam-lactim pairs, enamine-imine pairs, and annular forms wherea proton can occupy two or more positions of a heterocyclic system,e.g., 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and2H-isoindole and 1H- and 2H-pyrazole. Tautomeric forms can be inequilibrium or sterically locked into one form by appropriatesubstitution.

Compounds of the invention can also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.For example, isotopes of hydrogen include tritium and deuterium. One ormore constituent atoms of the compounds of the invention can be replacedor substituted with isotopes of the atoms in natural or non-naturalabundance. In some embodiments, the compound includes at least onedeuterium atom. For example, one or more hydrogen atoms in a compound ofthe present disclosure can be replaced or substituted by deuterium. Insome embodiments, the compound includes two or more deuterium atoms. Insome embodiments, the compound includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11 or 12 deuterium atoms. Synthetic methods for including isotopes intoorganic compounds are known in the art (Deuterium Labeling in OrganicChemistry by Alan F. Thomas (New York, N.Y., Appleton-Century-Crofts,1971; The Renaissance of H/D Exchange by Jens Atzrodt, Volker Derdau,Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007,7744-7765; The Organic Chemistry of Isotopic Labelling by James R.Hanson, Royal Society of Chemistry, 2011). Isotopically labeledcompounds can used in various studies such as NMR spectroscopy,metabolism experiments, and/or assays.

Substitution with heavier isotopes such as deuterium, may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample, increased in vivo half-life or reduced dosage requirements, andhence may be preferred in some circumstances. (A. Kerekes et. al. J.Med. Chem. 2011, 54, 201-210; R. Xu et. al. J. Label Compd. Radiopharm.2015, 58, 308-312).

The term, “compound,” as used herein is meant to include allstereoisomers, geometric isomers, tautomers and isotopes of thestructures depicted. The term is also meant to refer to compounds of theinventions, regardless of how they are prepared, e.g., synthetically,through biological process (e.g., metabolism or enzyme conversion), or acombination thereof.

All compounds, and pharmaceutically acceptable salts thereof, can befound together with other substances such as water and solvents (e.g.,hydrates and solvates) or can be isolated. When in the solid state, thecompounds described herein and salts thereof may occur in various formsand may, e.g., take the form of solvates, including hydrates. Thecompounds may be in any solid state form, such as a polymorph orsolvate, so unless clearly indicated otherwise, reference in thespecification to compounds and salts thereof should be understood asencompassing any solid state form of the compound.

In some embodiments, the compounds of the invention, or salts thereof,are substantially isolated. By “substantially isolated” is meant thatthe compound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, e.g., a composition enriched in the compounds of the invention.Substantial separation can include compositions containing at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 95%, at least about 97%, or at leastabout 99% by weight of the compounds of the invention, or salt thereof.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The expressions, “ambient temperature” and “room temperature,” as usedherein, are understood in the art, and refer generally to a temperature,e.g., a reaction temperature, that is about the temperature of the roomin which the reaction is carried out, e.g., a temperature from about 20°C. to about 30° C.

The present invention also includes pharmaceutically acceptable salts ofthe compounds described herein. The term “pharmaceutically acceptablesalts” refers to derivatives of the disclosed compounds wherein theparent compound is modified by converting an existing acid or basemoiety to its salt form. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, mineral or organic acid salts of basicresidues such as amines; alkali or organic salts of acidic residues suchas carboxylic acids; and the like. The pharmaceutically acceptable saltsof the present invention include the non-toxic salts of the parentcompound formed, e.g., from non-toxic inorganic or organic acids. Thepharmaceutically acceptable salts of the present invention can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, alcohols (e.g., methanol, ethanol,iso-propanol or butanol) or acetonitrile (MeCN) are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17^(th)Ed., (Mack Publishing Company, Easton, 1985), p. 1418, Berge et al., J.Pharm. Sci., 1977, 66(1), 1-19 and in Stahl et al., Handbook ofPharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002). Insome embodiments, the compounds described herein include the N-oxideforms.

Synthesis

Compounds of the invention, including salts thereof, can be preparedusing known organic synthesis techniques and can be synthesizedaccording to any of numerous possible synthetic routes, such as those inthe Schemes below.

The reactions for preparing compounds of the invention can be carriedout in suitable solvents which can be readily selected by one of skillin the art of organic synthesis. Suitable solvents can be substantiallynon-reactive with the starting materials (reactants), the intermediatesor products at the temperatures at which the reactions are carried out,e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected by the skilled artisan.

Preparation of compounds of the invention can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups is described, e.g., in Kocienski, Protecting Groups,(Thieme, 2007); Robertson, Protecting Group Chemistry, (OxfordUniversity Press, 2000); Smith et al., March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, 6^(th) Ed. (Wiley,2007); Peturssion et al., “Protecting Groups in Carbohydrate Chemistry,”J. Chem. Educ., 1997, 74(11), 1297; and Wuts et al., Protective Groupsin Organic Synthesis, 4th Ed., (Wiley, 2006).

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), massspectrometry or by chromatographic methods such as high performanceliquid chromatography (HPLC) or thin layer chromatography (TLC).

The Schemes below provide general guidance in connection with preparingthe compounds of the invention. One skilled in the art would understandthat the preparations shown in the Schemes can be modified or optimizedusing general knowledge of organic chemistry to prepare variouscompounds of the invention.

Compounds of Formula (I) can be prepared, e.g., using a process asillustrated in the schemes below.

A small molecule microtubule targeting moiety which contains an inherentthiol (R²—SH), such as DM1 or DM4, can be activated by the formation ofpyridyl disulfide (wherein X is, for example, H, halo, etc.), which canbe displaced with a thiolcontaining R¹ peptide in a disulfide exchangereaction to give the desired conjugate where —S—S— is the linking moietyL.

Alternatively, the R¹ peptide, which has an inherent thiol, can beactivated by the formation of pyridyl disulfide (II) which can bedisplaced with a thiol-containing R² in a disulfide exchange reaction togive the desired direct conjugate where L is —S—S—.

A protected ethyl amine, containing a thiol group that has beenactivated as pyridyl disulfide V can be reacted with thiol-containing R²in a disulfide exchange reaction to give VII. Disulfide VII can bedeprotected to give VIII and further reacted with a propionic maleimideIX in an acid coupling reaction to provide amide X. Amide X can bereacted with a thiol containing peptide in a Michael addition to givethe desired conjugate.

A thiol-containing butyric acid that has been activated as a pyridyldisulfide XI can be reacted with thiol containing R² in a disulfideexchange reaction to give XII. Disulfide acid XII can be reacted withethylaminomaleimide XIII inan acid coupling reaction to provide amideXIV. Amide XIV can be reacted with a thiol-containing peptide in aMichael addition to give the desired conjugate.

The peptides R¹ may be prepared using the solid-phase synthetic methodfirst described by Merrifield in J.A.C.S., Vol. 85, pgs. 2149-2154(1963), although other art-known methods may also be employed. TheMerrifield technique is well understood and is a common method forpreparation of peptides. Useful techniques for solid-phase peptidesynthesis are described in several books such as the text “Principles ofPeptide Synthesis” by Bodanszky, Springer Verlag 1984. This method ofsynthesis involves the stepwise addition of protected amino acids to agrowing peptide chain which was bound by covalent bonds to a solid resinparticle. By this procedure, reagents and by-products are removed byfiltration, thus eliminating the necessity of purifying intermediates.The general concept of this method depends on attachment of the firstamino acid of the chain to a solid polymer by a covalent bond, followedby the addition of the succeeding protected amino acids, one at a time,in a stepwise manner until the desired sequence is assembled. Finally,the protected peptide is removed from the solid resin support and theprotecting groups are cleaved off.

The amino acids may be attached to any suitable polymer. The polymermust be insoluble in the solvents used, must have a stable physical formpermitting ready filtration, and must contain a functional group towhich the first protected amino acid can be firmly linked by a covalentbond. Various polymers are suitable for this purpose, such as cellulose,polyvinyl alcohol, polymethylmethacrylate, and polystyrene.

Methods of Use

Provided herein is the use of the compounds of formula (I) in thetreatment of diseases, such as cancer or neurodegenerative disease.Another aspect of the present invention is the use of the compounds offormula (I) in the treatment of diseases involving acidic or hypoxicdiseased tissue, such as cancer. Hypoxia and acidosis are physiologicalmarkers of many disease processes, including cancer. In cancer, hypoxiais one mechanism responsible for development of an acid environmentwithin solid tumors. As a result, hydrogen ions must be removed from thecell (e.g., by a proton pump) to maintain a normal pH within the cell.As a consequence of this export of hydrogen ions, cancer cells have anincreased pH gradient across the cell membrane lipid bilayer and a lowerpH in the extracellular milieu when compared to normal cells. Oneapproach to improving the efficacy and therapeutic index of cytotoxicagents is to leverage this physiological characteristic to affordselective delivery of compound to hypoxic cells over healthy tissue.

In these methods of treatment, a therapeutically-effective amount of acompound of formula (I) or a pharmaceutically-acceptable salt thereofmay be administered as a single agent or in combination with other formsof therapy, such as ionizing radiation or cytotoxic agents in the caseof cancer. In combination therapy, the compound of formula (I) may beadministered before, at the same time as, or after the other therapeuticmodality, as will be appreciated by those of skill in the art. Eithermethod of treatment (single agent or combination with other forms oftherapy) may be administered as a course of treatment involving multipledoses or treatments over a period of time.

Examples of cancers that are treatable using the compounds of thepresent disclosure include, but are not limited to, colorectal cancer,gastric cancer, bone cancer, pancreatic cancer, skin cancer, cancer ofthe head or neck, cutaneous or intraocular malignant melanoma, uterinecancer, ovarian cancer, rectal cancer, cancer of the anal region,stomach cancer, testicular cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, endometrial cancer,carcinoma of the cervix, carcinoma of the vagina, carcinoma of thevulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of theesophagus, cancer of the small intestine, cancer of the endocrinesystem, cancer of the thyroid gland, cancer of the parathyroid gland,cancer of the adrenal gland, sarcoma of soft tissue, cancer of theurethra, cancer of the penis, chronic or acute leukemias including acutemyeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, solid tumors of childhood,lymphocytic lymphoma, cancer of the bladder, cancer of the kidney orurethra, carcinoma of the renal pelvis, neoplasm of the central nervoussystem (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axistumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma,epidermoid cancer, squamous cell cancer, T-cell lymphoma,environmentally induced cancers including those induced by asbestos, andcombinations of said cancers.

In some embodiments, cancers treatable with compounds of the presentdisclosure include bladder cancer, bone cancer, glioma, breast cancer(e.g., triple-negative breast cancer), cervical cancer, colon cancer,colorectal cancer, endometrial cancer, epithelial cancer, esophagealcancer, Ewing's sarcoma, pancreatic cancer, gallbladder cancer, gastriccancer, gastrointestinal tumors, head and neck cancer (upperaerodigestive cancer), intestinal cancers, Kaposi's sarcoma, kidneycancer, laryngeal cancer, liver cancer (e.g., hepatocellular carcinoma),lung cancer (e.g., non-small cell lung cancer, adenocarcinoma),melanoma, prostate cancer, rectal cancer, renal clear cell carcinoma,skin cancer, stomach cancer, testicular cancer, thyroid cancer, anduterine cancer.

In some embodiments, cancers treatable with compounds of the presentdisclosure include melanoma (e.g., metastatic malignant melanoma), renalcancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormonerefractory prostate adenocarcinoma), breast cancer, triple-negativebreast cancer, colon cancer and lung cancer (e.g. non-small cell lungcancer and small cell lung cancer). Additionally, the disclosureincludes refractory or recurrent malignancies whose growth may beinhibited using the compounds of the disclosure.

In some embodiments, cancers that are treatable using the compounds ofthe present disclosure include, but are not limited to, solid tumors(e.g., prostate cancer, colon cancer, esophageal cancer, endometrialcancer, ovarian cancer, uterine cancer, renal cancer, hepatic cancer,pancreatic cancer, gastric cancer, breast cancer, lung cancer, cancersof the head and neck, thyroid cancer, glioblastoma, sarcoma, bladdercancer, etc.), hematological cancers (e.g., lymphoma, leukemia such asacute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML),chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML),DLBCL, mantle cell lymphoma, Non-Hodgkin lymphoma (including relapsed orrefractory NHL and recurrent follicular), Hodgkin lymphoma or multiplemyeloma) and combinations of said cancers.

In certain embodiments, a compound of formula (I) or apharmaceutically-acceptable salt thereof may be used in combination witha chemotherapeutic agent, a targeted cancer therapy, an immunotherapy orradiation therapy. The agents can be combined with the present compoundsin a single dosage form, or the agents can be administeredsimultaneously or sequentially as separate dosage forms. In someembodiments, the chemotherapeutic agent, targeted cancer therapy,immunotherapy or radiation therapy is less toxic to the patient, such asby showing reduced bone marrow toxicity, when administered together witha compound of formula (I), or a pharmaceutically acceptable saltthereof, as compared with when administered in combination with thecorresponding microtubule targeting agent (e.g., R²—H).

Suitable chemotherapeutic or other anti-cancer agents include, forexample, alkylating agents (including, without limitation, nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas andtriazenes) such as uracil mustard, chlormethine, cyclophosphamide(Cytoxan™), ifosfamide, melphalan, chlorambucil, pipobroman,triethylene-melamine, triethylenethiophosphoramine, busulfan,carmustine, lomustine, streptozocin, dacarbazine, and temozolomide.

Other suitable agents for use in combination with the compounds of thepresent invention include: dacarbazine (DTIC), optionally, along withother chemotherapy drugs such as carmustine (BCNU) and cisplatin; the“Dartmouth regimen,” which consists of DTIC, BCNU, cisplatin andtamoxifen; a combination of cisplatin, vinblastine, and DTIC; ortemozolomide. Compounds according to the invention may also be combinedwith immunotherapy drugs, including cytokines such as interferon alpha,interleukin 2, and tumor necrosis factor (TNF).

Suitable chemotherapeutic or other anti-cancer agents include, forexample, antimetabolites (including, without limitation, folic acidantagonists, pyrimidine analogs, purine analogs and adenosine deaminaseinhibitors) such as methotrexate, 5-fluorouracil, floxuridine,cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate,pentostatine, and gemcitabine.

Suitable chemotherapeutic or other anti-cancer agents further include,for example, certain natural products and their derivatives (forexample, vinca alkaloids, antitumor antibiotics, enzymes, lymphokinesand epipodophyllotoxins) such as vinblastine, vincristine, vindesine,bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin,idarubicin, ara-C, paclitaxel (TAXOL™), mithramycin, deoxycoformycin,mitomycin-C, L-asparaginase, interferons (especially IFN-α), etoposide,and teniposide.

Other cytotoxic agents that can be administered in combination with thecompounds of the invention include, for example, navelbene, CPT-11,anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,ifosamide, and droloxafine.

Also suitable are cytotoxic agents such as, for example,epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor;procarbazine; mitoxantrone; platinum coordination complexes such ascis-platin and carboplatin; biological response modifiers; growthinhibitors; antihormonal therapeutic agents; leucovorin; tegafur; andhaematopoietic growth factors.

Other anti-cancer agent(s) include antibody therapeutics such astrastuzumab (Herceptin), antibodies to costimulatory molecules such asCTLA-4, 4-1BB and PD-1, or antibodies to cytokines (IL-10, TGF-α, etc.).

Other anti-cancer agents also include those that block immune cellmigration such as antagonists to chemokine receptors, including CCR2 andCCR4.

Other anti-cancer agents also include those that augment the immunesystem such as adjuvants or adoptive T cell transfer.

Anti-cancer vaccines that can be administered in combination with thecompounds of the invention include, for example, dendritic cells,synthetic peptides, DNA vaccines and recombinant viruses.

Other suitable agents for use in combination with the compounds of thepresent invention include chemotherapy combinations such asplatinum-based doublets used in lung cancer and other solid tumors(cisplatin or carboplatin plus gemcitabine; cisplatin or carboplatinplus docetaxel; cisplatin or carboplatin plus paclitaxel; cisplatin orcarboplatin plus pemetrexed) or gemcitabine plus paclitaxel boundparticles (Abraxane®).

Compounds of this invention may be effective in combination withanti-hormonal agents for treatment of breast cancer and other tumors.Suitable examples are anti-estrogen agents including but not limited totamoxifen and toremifene, aromatase inhibitors including but not limitedto letrozole, anastrozole, and exemestane, adrenocorticosteroids (e.g.prednisone), progestins (e.g. megastrol acetate), and estrogen receptorantagonists (e.g. fulvestrant). Suitable anti-hormone agents used fortreatment of prostate and other cancers may also be combined withcompounds of the present invention. These include anti-androgensincluding but not limited to flutamide, bicalutamide, and nilutamide,luteinizing hormone-releasing hormone (LHRH) analogs includingleuprolide, goserelin, triptorelin, and histrelin, LHRH antagonists(e.g. degarelix), androgen receptor blockers (e.g. enzalutamide) andagents that inhibit androgen production (e.g. abiraterone).

Compounds of the present invention may be combined with or administeredin sequence with other agents against membrane receptor kinasesespecially for patients who have developed primary or acquiredresistance to the targeted therapy. These therapeutic agents includeinhibitors or antibodies against EGFR, Her2, VEGFR, c-Met, Ret, IGFR1,or Flt-3 and against cancer-associated fusion protein kinases such asBcr-Abl and EML4-Alk. Inhibitors against EGFR include gefitinib anderlotinib, and inhibitors against EGFR/Her2 include but are not limitedto dacomitinib, afatinib, lapitinib and neratinib. Antibodies againstthe EGFR include but are not limited to cetuximab, panitumumab andnecitumumab. Inhibitors of c-Met may be used in combination with thecompounds of the invention. These include onartumzumab, tivantnib, andINC-280. Agents against Abl (or Bcr-Abl) include imatinib, dasatinib,nilotinib, and ponatinib and those against Alk (or EML4-ALK) includecrizotinib.

Angiogenesis inhibitors may be efficacious in some tumors in combinationwith compounds of the invention. These include antibodies against VEGFor VEGFR or kinase inhibitors of VEGFR. Antibodies or other therapeuticproteins against VEGF include bevacizumab and aflibercept. Inhibitors ofVEGFR kinases and other anti-angiogenesis inhibitors include but are notlimited to sunitinib, sorafenib, axitinib, cediranib, pazopanib,regorafenib, brivanib, and vandetanib

Activation of intracellular signaling pathways is frequent in cancer,and agents targeting components of these pathways have been combinedwith receptor targeting agents to enhance efficacy and reduceresistance. Examples of agents that may be combined with compounds ofthe present invention include inhibitors of the PI3K-AKT-mTOR pathway,inhibitors of the Raf-MAPK pathway, inhibitors of JAK-STAT pathway, andinhibitors of protein chaperones and cell cycle progression.

Agents against the PI3 kinase include but are not limited topilaralisib,idelalisib, buparlisib. Inhibitors of mTOR such as rapamycin, sirolimus,temsirolimus, and everolimus may be combined with compounds of theinvention. Other suitable examples include but are not limited tovemurafenib and dabrafenib (Raf inhibitors) and trametinib, selumetiniband GDC-0973 (MEK inhibitors). Inhibitors of one or more JAKs (e.g.,ruxolitinib, baricitinib, tofacitinib), Hsp90 (e.g., tanespimycin),cyclin dependent kinases (e.g., palbociclib), HDACs (e.g.,panobinostat), PARP (e.g., olaparib), and proteasomes (e.g., bortezomib,carfilzomib) can also be combined with compounds of the presentinvention. A further example of a PARP inhibitor that can be combinedwith a compound of the invention is talazoparib.

Methods for the safe and effective administration of most of thesechemotherapeutic agents are known to those skilled in the art. Inaddition, their administration is described in the standard literature.For example, the administration of many of the chemotherapeutic agentsis described in the “Physicians' Desk Reference” (PDR, e.g., 1996edition, Medical Economics Company, Montvale, N.J.), the disclosure ofwhich is incorporated herein by reference as if set forth in itsentirety.

The phrase “therapeutically effective amount” of a compound (therapeuticagent, active ingredient, drug, etc.) refers to an amount of thecompound to be administered to a subject in need of therapy or treatmentwhich alleviates a symptom, ameliorates a condition, or slows the onsetof disease conditions, according to clinically acceptable standards forthe disorder or condition to be treated. For instance, a therapeuticallyeffective amount can be an amount which has been demonstrated to have adesired therapeutic effect in an in vitro assay, an in vivo animalassay, or a clinical trial. The therapeutically effective amount canvary based on the particular dosage form, method of administration,treatment protocol, specific disease or condition to be treated, thebenefit/risk ratio, etc., among numerous other factors.

Said therapeutically effective amount can be obtained from a clinicaltrial, an animal model, or an in vitro cell culture assay. It is knownin the art that the effective amount suitable for human use can becalculated from the effective amount determined from an animal model oran in vitro cell culture assay. For instance, as reported by Reagan-Shawet al., FASEB J. 2008: 22(3) 659-61, “μg/ml” (effective amount based onin vitro cell culture assays)=“mg/kg body weight/day” (effective amountfor a mouse). Furthermore, the effective amount for a human can becalculated from the effective amount for a mouse based on the fact thatthe metabolism rate of mice is 6 times faster than that of humans.

As an example of treatment using a compound of formula (I) incombination with a cytotoxic agent, a therapeutically-effective amountof a compound of formula (I) may be administered to a patient sufferingfrom cancer as part of a treatment regimen also involving atherapeutically-effective amount of ionizing radiation or a cytotoxicagent. In the context of this treatment regimen, the term“therapeutically-effective” amount should be understood to meaneffective in the combination therapy. It will be understood by those ofskill in the cancer-treatment field how to adjust the dosages to achievethe optimum therapeutic outcome.

Similarly, the appropriate dosages of the compounds of the invention fortreatment of non-cancerous diseases or conditions (such ascardiovascular diseases) may readily be determined by those of skill inthe medical arts.

The term “treating” as used herein includes the administration of acompound or composition which reduces the frequency of, delays the onsetof, or reduces the progression of symptoms of a disease involving acidicor hypoxic diseased tissue, such as cancer, stroke, myocardialinfarction, or long-term neurodegenerative disease, in a subjectrelative to a subject not receiving the compound or composition. Thiscan include reversing, reducing, or arresting the symptoms, clinicalsigns, or underlying pathology of a condition in a manner to improve orstabilize a subject's condition (e.g., regression of tumor growth, forcancer or decreasing or ameliorating myocardial ischemia reperfusioninjury in myocardial infarction, stroke, or the like cardiovasculardisease). The terms “inhibiting” or “reducing” are used for cancer inreference to methods to inhibit or to reduce tumor growth (e.g.,decrease the size of a tumor) in a population as compared to anuntreated control population.

All publications (including patents) mentioned herein are incorporatedherein by reference for the purpose of describing and disclosing, forexample, the constructs and methodologies that are described in thepublications, which might be used in connection with the disclosureherein described. The publications discussed throughout the text areprovided solely for their disclosure prior to the filing date of thepresent application.

Disclosed herein are several types of ranges. When a range of any typeis disclosed or claimed, the intent is to disclose or claim individuallyeach possible number that such a range could reasonably encompass,including end points of the range as well as any sub-ranges andcombinations of sub-ranges encompassed therein. When a range oftherapeutically effective amounts of an active ingredient is disclosedor claimed, for instance, the intent is to disclose or claimindividually every possible number that such a range could encompass,consistent with the disclosure herein. For example, by a disclosure thatthe therapeutically effective amount of a compound can be in a rangefrom about 1 mg/kg to about 50 mg/kg (of body weight of the subject).

Formulation, Dosage Forms and Administration

To prepare the pharmaceutical compositions of the present invention, acompound of Formula (I) or a pharmaceutically-acceptable salt thereof iscombined as the active ingredient in intimate admixture with apharmaceutical carrier according to conventional pharmaceuticalcompounding techniques, which carrier may take a wide variety of formsdepending on the form of preparation desired for administration, e.g.,oral or parenteral. In preparing the compositions in oral dosage form,any of the usual pharmaceutical media may be employed, such as forexample, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, and the like in the case of oral liquidpreparations such as for example, suspensions, elixirs, and solutions;or carriers such as starches, sugars, diluents, granulating agents,lubricants, binders, disintegrating agents, and the like in a case oforal solid preparations, such as for example, powders, capsules, andtablets. Because of their ease in administration, tablets and capsulesrepresent the most advantageous oral dosage unit form, in which casesolid pharmaceutical carriers are obviously employed. If desired,tablets may be sugar coated or enteric coated by standard techniques.For parenterals, the carrier will usually comprise sterile water,although other ingredients, for example, to aid solubility or forpreservative purposes, may be included. Injectable suspensions may alsobe prepared, in which case appropriate liquid carriers, suspendingagents, and the like may be employed. One of skill in the pharmaceuticaland medical arts will be able to readily determine a suitable dosage ofthe pharmaceutical compositions of the invention for the particulardisease or condition to be treated.

EXAMPLES

As used herein, all abbreviations, symbols and conventions areconsistent with those used in the contemporary scientific literature.See, e.g., Janet S. Dodd, ed., The ACS Style Guide: A Manual for Authorsand Editors, 2nd Ed., Washington, D.C.: American Chemical Society, 1997.The following definitions describe terms and abbreviations used herein:

-   -   Brine: a saturated NaCl solution in water    -   DCM: dichloromethane    -   TFA: trifluoroacetic acid    -   DIPEA: diisopropylethylamine    -   DMA: dimethylacetamide    -   DME: dimethoxyethane    -   DMF: dimethylformamide    -   DMSO: methylsulfoxide    -   DTT: dithiothreitol    -   MSD: mass spec detector    -   Et₂O: ethyl ether    -   EtOAc: ethyl acetate    -   EtOH: ethyl alcohol    -   HATU: O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    -   hexafluorophosphate    -   HOBt: 1-hydroxybenzotriazole    -   RP: reverse phase    -   HPLC: high performance liquid chromatography    -   IPA: isopropanol    -   LAH: lithium aluminum hydride    -   N-BuLi: n-butyl lithium    -   LC-MS: liquid chromatography-mass spectrometry    -   LDA: lithium diisoproylethylamide    -   Me: methyl    -   MeOH: methanol    -   MTBE: methyl t-butyl ether    -   NMP: N-methylpyrrolidine    -   Ph: phenyl    -   PNPC: para-nitrophenylchloroformate    -   RT or rt: room temperature    -   SFC: supercritical fluid chromatography    -   TBA: tetrabutylammonium iodide    -   TBME: tert-butylmethyl ether    -   tBu: tertiary butyl    -   THF: tetrahydrofuran    -   TEA: triethylamine    -   TMEDA: tetramethylethylenediamine    -   GSH: Glutathione    -   GS: Glutathione bonded at sulfur    -   LiOH: lithium hydroxide    -   DPPA: diphenyl phosphoryl azide    -   Sn(Bu)₂(Laurate)₂: dibutyltin dilaurate    -   PBS: phosphate buffered saline    -   ACN: acetonitrile    -   AcOH: acetic acid    -   EEDQ: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline    -   DMAP: 4-dimethylaminopyridine    -   EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide        The HPLC methods employed are set forth below.

HPLC Methods

A: Sunfire C18 150×4.6 mm; H₂O/Acetonitrile w/TFA modifier (0.05%); Flowrate: 1 ml/min; Wavelength=217 nM.

B: Ace Equivalence 250×4.6 mm; H₂O/Acetonitrile w/TFA modifier (0.05%);Flow rate: 1 ml/min; Wavelength=217 nM.

C: Sunfire C18 150×30 mm; H₂O/Acetonitrile w/TFA modifier (0.05%); Flowrate: 30 ml/min; Wavelength=217 nM.

D: Sunfire C18 150×4.6 mm; H₂O/Acetonitrile w/AcOH modifier (0.5%); Flowrate: 1 ml/min; Wavelength=217 nM

E: Sunfire C18 150×30 mm; H₂O/Acetonitrile w/AcOH modifier (0.5%); Flowrate: 30 ml/min; Wavelength=217 nM.

F: Agilent 1100/1200/1260 or 1290 systems (coupled or uncoupled withMS).

HPLC Parameters Mobile Phase A 0.1% AcOH in water Mobile Phase B 0.1%AcOH in ACN Column Merck Chromolith RP-18e Column Temperature rtAutosampler rt Temperature Injection Volume 5 μL Flow Rate 1 mL/minuteWavelength Agilent diode array detector at λ = 254, 220 or 280 nmGradient Program Time (min) % A % B Initial 95.00  5.00 4.00  5.00 95.004.99  5.00 95.00 5.00 95.00  5.00 6.00 95.00  5.00 Run Time 6.00G: Agilent 1100/1200/1260 or 1290 systems (coupled or uncoupled withMS).

HPLC Parameters Mobile Phase A 0.1% TFA in water Mobile Phase B 0.1% TFAin ACN Column Agilent Eclipse XDB C8 column (3.5 μm, 4.6 × 150 mm)Column Temperature 40° C. Autosampler rt Temperature Injection Volume 5μL Flow Rate 1.5 mL/minute Wavelength Agilent diode array detector at λ= 254, 220 or 280 nm Gradient Program Time (min) % A % B Initial 80.00 20.00  0.20 80.00  20.00  7.50 20.00  80.00  8.00  0.00 100.00  9.00 0.00 100.00 10.00 80.00  20.00 Run Time 10.00

Mass Spectrometry Methods

Maldi-TOF (Matrix-assisted laser desorption/ionization-Time of Flight)mass spectrometry was measured on an Applied Biosystems Voyager System6268. The sample was prepared as a matrix of α-cyano hydroxy cinnamicacid on an AB Science plate (Part #V700666).

ESI (Electrospray Ionization) mass spectrometry was measured on eitheran Agilent 1100 series LC-MS with a 1946 MSD or a Waters Xevo Qtofhigh-resolution MS, both providing a mass/charge species (m/z=3).

The source of the starting materials employed in the Examples are setforth below in the following tables.

TABLE 2 Starting materials for R² Synthesis, Reference or R² Code R²HStructure Purchased R²SH-1

MedKoo 123212 R²SH-2

MedKoo 206839 R²SH-3

US 20040235840 A1Synthesis of Intermediate I (R²S—S-Pyr)

To[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[methyl(3-sulfanylpropanoyl)amino]propanoate (46.7 mg, 0.06 mmol)in 1 mL of CH₃CN was added 2-(2-pyridyldisulfanyl)pyridine (20.0 mg,0.09 mmol). The mixture was concentrated and purified (SiO₂, 0-10%MeOH/CH₂Cl₂) to give[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[methyl-[3-(2-pyridyldisulfanyl)propanoyl]amino]propanoate (53.6mg, yield: 100%). MS m/z 847.1 [M+H]⁺.

Synthesis of Pv3-S-Pyr (Intermediate II-3)

Pv3 (250 mg, 0.06 mmol; as a free flowing solid) and2-(2-pyridyldisulfanyl)pyridine (0.110 g, 0.5 mol) were dissolved inMeOH (10 mL) and the reaction stirred overnight at room temperature.LC-MS indicates the desired product was formed. The reaction mixture wasconcentrated and the residue taken up in DMSO and purified by reversephase column chromatography (40-65% CH₃CN/H₂O (0.5% AcOH), 13 min) togive 212 mg of the desired product (187 mg, yield: 74.9%). MS m/z=31273.4.

Intermediates II-1, II-2 and II-6 were prepared analogously to II-3,using Pv1, Pv2, and Pv6, as shown below:

MS A: Maldi-TOF Intermediate Structure B: m/z = 3 II-1 Pv1-SPyr B:1130.1 II-2 Pv2-SPyr B: 1373.9 II-3 Pv3-SPyr B: 1273.4 II-6 Pv6-SPyr B:1450.3

TABLE 3 Starting materials for L groups Purchased, ReferenceIntermediate Structure or Synthesized III-1

Enamine EN3000-33931 III-2

Astatech 39541 III-3

Enamine EN3000-6731388 III-4

Enamine EN3000-6731596 III-5

Astatech 39018Synthesis of Intermediate VI-2

1-Amino-2-methyl-propane-2-thiol hydrochloride (100 mg, 0.706 mmol) wasdissolved in CH₂Cl₂ (7 mL) and to it was added 9H-fluoren-9-ylmethylcarbonochloridate (274 mg, 1.06 mmol) and N,N-diisopropylethylamine (182mg, 1.41 mmol). The reaction mixture was stirred at room temperatureovernight. The reaction mixture was washed with water and concentrated.The residue was purified by column chromatography (0-50% EtOAc/hexanes)to give 9H-fluoren-9-ylmethyl N-(2-methyl-2-sulfanyl-propyl)carbamate(213 mg, yield: 92.2%). MS m/z 350.1 [M+Na]⁺.

Synthesis of Intermediate V-1

2-(2-Pyridyldisulfanyl)pyridine (746 mg, 3.38 mmol) was dissolved inMeOH (15 mL) and to it was added tert-butyl N-(2-sulfanylethyl)carbamate(200 mg, 1.13 mmol). The reaction was stirred for 3 h at roomtemperature. The mixture was concentrated and the residue purified bycolumn chromatography (0-50% EtOAc/hexanes) to give tert-butylN-[2-(2-pyridyldisulfanyl)ethyl]carbamate (200 mg, yield: 61.9%). MS m/z287.1 [M+H]⁺.

Synthesis of Intermediate V-2

2-(2-Pyridyldisulfanyl)ethanamine hydrochloride (200 mg, 0.898 mmol) wasdissolved in CH₂Cl₂ and to it was added 9H-fluoren-9-ylmethylcarbonochloridate (348 mg, 1.35 mmol) and N,N-diisopropylethylamine (232mg, 1.80 mmol). The reaction mixture was stirred at RT for 2 h, washedwith water and concentrated. The residue was purified by columnchromatography (0-50% EtOAc/hexanes) to give 9H-fluoren-9-ylmethylN-[2-(2-pyridyldisulfanyl)ethyl]carbamate (288 mg, yield: 78.5%). MS m/z409.1 [M+H]⁺.

Synthesis of Intermediate VII-1

To a vial containing[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[methyl(3-sulfanylpropanoyl)amino]propanoate (25.0 mg, 0.03 mmol)in 1 mL of CH₃CN was added tert-butylN-[2-(2-pyridyldisulfanyl)ethyl]carbamate (Intermediate V-1, 14.5 mg,0.051 mmol) and 4-methylmorpholine (0.138 mL, 1.25 mmol). The mixturewas stirred for 16 h. LC-MS analysis indicates the desired material. Themixture was concentrated, dissolved in 50 mL of EtOAc and washed with1×25 mL of sat. NH₄Cl and 1×25 mL of sat. brine. The organic phase wasdried with MgSO₄, filtered and concentrated. The crude residue waspurified (SiO₂, 0-100% EtOAc/hexanes) to give[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[3-[2-(tert-butoxycarbonylamino)ethyldisulfanyl]propanoyl-methyl-amino]propanoate(30.9 mg, yield: 100%). MS m/z 913.2 [M+H]⁺.

Synthesis of Intermediate VII-2

Intermediate VII-2 was prepared analogously to VII-1, using IntermediateV-2 in place of Intermediate V-1.

Synthesis of Intermediate VII-3

To a vial containing[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[3-[2-(9H-fluoren-9-ylmethoxycarbonylamino)ethyldisulfanyl]propanoyl-methyl-amino]propanoate(25.0 mg, 0.03 mmol) in 1 mL of CH₃CN was added 9H-fluoren-9-ylmethylN-(2-methyl-2-sulfanyl-propyl)carbamate (14.5 mg, 0.044 mmol) and4-methylmorpholine (0.120 mL, 1.09 mmol). The mixture was stirred for 16h. LC-MS analysis indicated the desired material was formed. The mixturewas concentrated, dissolved in 50 mL of EtOAc and washed with 1×25 mL ofsat. NH₄Cl and 1×25 mL of sat. brine. The organic phase was dried withMgSO₄, filtered and concentrated. The crude residue was purified (SiO₂,0-100% EtOAc/hexanes) to [(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[3-[[2-(9H-fluoren-9-ylmethoxycarbonylamino)-1,1-dimethyl-ethyl]disulfanyl]propanoyl-methyl-amino]propanoate(0.0313 g, yield: 100%). MS m/z 1085.0 [M+Na]*.

Synthesis of Intermediate VIII-1 (BOC Deprotection)

[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[3-[2-(tert-butoxycarbonylamino)ethyldisulfanyl]propanoyl-methyl-amino]propanoate(31.9 mg, 0.05 mmol) was dissolved in 0.3/0.1/0.1 mL of CH₃CN/H₂O/TFA.The mixture was stirred for 36 h. LC-MS indicated complete deprotection.The mixture was purified by prep HPLC (20-95% CH₃CN/H₂O w/ 0.05% TFA) togive[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[3-(2-aminoethyldisulfanyl)propanoyl-methyl-amino]propanoate;2,2,2-trifluoroacetic acid (22.9 mg, yield: 70.7%). MS m/z 813.2 [M+H]⁺.

Alternative Synthesis of Intermediate VIII-1 (FMOC Deprotection)

To a vial containing[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[3-[2-(9H-fluoren-9-ylmethoxycarbonylamino)ethyldisulfanyl]propanoyl-methyl-amino]propanoate(Intermediate VII-2; 29.6 mg, 0.03 mmol) was added 0.5 mL of DMF and4-methylmorpholine (0.120 mL, 1.09 mmol). The mixture was heated for 16h at 40° C. LC-MS confirmed complete deprotection. The mixture waspurified by (20-95% CH₃CN/H₂O w/ 0.05% TFA) to give[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[3-(2-aminoethyldisulfanyl)propanoyl-methyl-amino]propanoatetrifluoroacetate (22.9 mg, yield: 86.4%). MS m/z 813.2 [M+H]⁺.

Synthesis of Intermediate VIII-2

Intermediate VIII-2 was prepared analogously to Intermediate VIII-1. MSm/z 841.2 [M+H]⁺.

Synthesis of Intermediate X-1

To[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[3-(2-aminoethyldisulfanyl)propanoyl-methyl-amino]propanoatetrifluoroacetate (Intermediate VIII-1; 45.8 mg, 0.05 mmol) in 1 mL ofDMF was added 3-(2,5-dioxopyrrol-1-yl)propanoic acid (12.5 mg, 0.074mmol), TBTU (23.8 g, 0.074 mmol) and DIPEA (0.0169 mL, 0.1 mmol). LC-MSindicated complete conversion to the product. The mixture was dilutedwith 50 mL of EtOAc. This was washed with 1×25 mL sat NH₄Cl, 4×25 mL H₂Oand 1×25 mL of H₂O. The organic phase was dried with MgSO₄, filtered andconcentrated. The crude product was purified (SiO₂, 0-10% MeOH/CL₂Cl₂)to give[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethyldisulfanyl]propanoyl-methyl-amino]propanoate(17.3 mg, yield: 36.5%) MS m/z 986.1 [M+Na]*.

Example 2: Synthesis of Compound 2

To a vial containing Pv2 (25.0 mg, 0.006 mmol; as a free flowing solid)and[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[methyl-[3-(2-pyridyldisulfanyl)propanoyl]amino]propanoate (7.70mg, 0.009 mmol) was added 1 mL of degassed DMF and 0.5 mL degassed H₂O.To this was added CH₃CO₂H (0.0103 mL, 0.180 mmol). The mixture wasstirred for 72 h. LC-MS indicated formation of desired product. Themixture was purified by prep HPLC (Sunfire C18 150×30 mm; 20-77%H₂O/Acetonitrile w/0.5% AcOH modifier; 15 min run; Flow rate: 30 ml/min;Wavelength=217 nM) to give the desired conjugate (17.0 mg, yield:59.1%).

Example 6: Synthesis of Compound 6

To a vial containing Pv2-SPyr (Intermediate II-2; 27.0 mg, 6.55e-6 mol)and[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl](2S)-2-[methyl-(4-methyl-4-sulfanyl-pentanoyl)amino]propanoate (7.67 mg,0.01 mmol). To this was added 1 mL of degassed DMF and 0.5 mL degassedH₂O. To this was added CH₃CO₂H (0.015 mL, 0.262 mmol). The mixture wasstirred for 72 h. LC-MS indicated formation of desired product. Themixture was purified by prep HPLC (Sunfire C18 150×30 mm; 20-80%H₂O/Acetonitrile w/ 0.5% AcOH modifier; 16 min run; Flow rate: 30ml/min; Wavelength=217 nM) to give the desired conjugate (11.4 g, yield:36.6%).

Example 9. Synthesis of Compound 9

To a vial containing Pv2 (25.0 mg, 0.006 mol; as a free flowing solid)and[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10(26),11,13,16,18-pentaen-6-yl](2S)-2-[3-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethyldisulfanyl]propanoyl-methyl-amino]propanoate(Intermediate X-1; 0.00877 g, 0.01 mmol) was added 1 mL of CH₃CN. Themixture was heterogeneous. To this was added 0.5 mL of CH₃CN, 0.5 mL ofH₂O and 0.5 mL of MeOH. Homogeneity was not achieved. The mixture wasstirred rigorously for 72 h. LC-MS indicated formation of desiredproduct. The mixture was purified by prep HPLC (Sunfire C18 150×30 mm;45-61% H₂O/Acetonitrile w/0.05% TFA modifier; 13 min run; Flow rate: 30ml/min; Wavelength=217 nM) to give desired conjugate (21.1 mg, yield:70.0%).

Compounds 1, 3, and 4 were synthesized analogously to the compound ofCompound 2, using Pv1, Pv3, and Pv4, respectively. Compounds 5, 7, and 8were synthesized analogously to Compound 6, using Intermediates II-1,II-3, and II-6, respectively.

TABLE 4 Example Compounds MS A: Maldi- Conditions TOF (M+) % ACN/H₂OCom- B: ESI Run Time pound Structure (m/z = 3) RT 1

B: 1339.3 D 20-95% 11 min 7.28 min 2

B: 1582.7 A 20-95% 11 min 7.0 min 3

B: 1483.8 D 20-95% 11 min 7.82 min 4

B: 1659.4 A 20-95% 11 min 6.92 min 5

B: 1352.8 A 20-95% 11 min 6.81 min 6

B: 1597.0 A 20-95% 11 min 7.44 min 7

B: 1487.1 A 20-95% 11 min 6.60 min 8

B: 1673.4 A 20-95% 11 min 7.14 min 9

B: 1659.4 A 20-95% 11 min 7.16 min

Example 5: Detailed Synthesis of Compound 5

To Pv1 (50.0 mg, 1.48e-5 mol) and[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy-12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa-10,12,14(26),16,18-pentaen-6-yl](2S)-2-[methyl-(4-methyl-4-sulfanyl-pentanoyl)amino]propanoate (0.0150g, 1.92e-5 mol) in 3 mL of 2:1 CH₃CN/H₂O was added N-methylmorpholine(0.0600 mL, 0.000546 mol). The mixture was stirred for 36 h. LC-MSanalysis indicated formation of the desired material. The mixture waspurified by Gilson prep HPLC (Sunfire C18 3×10 mm; 20-80% CH₃CN/H₂O w/0.05% TFA; 16 min run; 13.5 min) to give desired conjugate. The mixturewas purified by Gilson prep HPLC (Sunfire C18 30×150 m; 20-72% CH₃CN/H₂Ow/ 0.05% TFA; 15 min run; 12.5 min; retention time: 6.847 min) to giveCompound 5 (0.0322 g, 7.94e-6 mol, yield: 53.8%). ESI (m/z=3): 1352.8.

Example 5a: Alternative Synthesis of Compound 5 Step 1. Preparation ofPv1-S-Pyridyl

Peptide Pv1 and 2,2′-dipyridyl disulfide were dissolved in MeOH and thereaction was stirred overnight. LC-MS indicated the desired product wasformed. The reaction mixture was concentrated and the residue was takenup in DMSO and purified by reverse phase column chromatography (40-75%ACN/H2O (0.5% AcOH), 15 min) to give 212 mg of the desired product.

Step 2. Preparation of Compound 5

To a vial containing Pv1-SPyr (25.0 mg, 736e-6 mol) and[(1S,2R,3S,6S,16E,18E,20R,21S)-11-chloro-21-hydroxy12,20-dimethoxy-2,5,9,16-tetramethyl-8,23-dioxo-4,24-dioxa-9,22-diazatetracyclo[19.3.1.110,14.03,5]hexacosa10,12,14(26),16,18-pentaen-6-yl](2S)-2-[methyl-(4-methyl-4-sulfanyl-pentanoyl)amino]propanoate (0.00864g, 1.11e-5 mol). To this mixture was added 1 mL of degassed DMF and 0.5mL degassed H₂O. CH₃CO₂H (0.017 mL, 0.000295 mol) was added. The mixturewas stirred for 72 h. LC-MS indicated formation of the desired product.The mixture was purified by Gilson prep HPLC (Sunfire C18 30×150 mm;20-80 CH3CN/H2O w/ 0.5% AcOH; 16 min run; 12.9 min) to give Compound 5(0.00750 g, 1.85e-6 mol, yield: 25.1%).

Example 10: Synthesis of Compound 10

Step 1.(1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-ylN-(4-((2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)thio)-4-methylpentanoyl)-N-methyl-L-alaninate

180 mg of DM4 (0.23 mmol) and 57 mg of bromoacetic acidN-hydroxysuccinimide ester (0.24 mmol) were dissolved in DMF (4.6 mL)and cooled in ice-water bath. 36.2 μL of DBU (0.24 mmol) was added atonce and the mixture was allowed to warm to RT. At that moment LC/MSindicated nearly 95% conversion and the reaction was quenched withaddition of 0.1 mL AcOH. Crude reaction mixture was directly loaded ontoa 50 g C18Aq column and purified via standard 10-100% B gradient (A:water w. 0.05% AcOH; B: water w. 0.05% AcOH). Product containingfractions were lyophilized to afford 160 mg of product (77% yield). HPLCpurity at 254 nm: 96%. Retention time: 2.83 min (Method F). LCMS: 935.4MH⁺.

Step 2.1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1+-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-ylN-(4-((2-((2-aminoethyl)amino)-2-oxoethyl)thio)-4-methylpentanoyl)-N-methyl-L-alaninate

25 mg of1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-33,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-ylN-(4-((2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)thio)-4-methylpentanoyl)-N-methyl-L-alaninate(0.027 mmol) and 36 mg ofN1-((4-methoxyphenyl)diphenylmethyl)ethane-1,2-diamine (0.11 mmol, 4 eq)were dissolved in dioxane (1 mL). After 3 h, the reaction appeared to becomplete on LC/MS. The mixture was concentrated to dryness and dissolvedin 80% AcOH in water (2 mL). LC/MS showed complete deprotection of theintermediate and the mixture was directly freeze-dried. The residue wasdissolved in DMSO (1 mL) and loaded onto 15.5 g C18Aq column andpurified via standard 5-100% B gradient (A: water w. 0.05% AcOH; B:water w. 0.05% AcOH). Product containing fractions were lyophilized toafford 18 mg of product. HPLC purity at 254 nm: 95%. Retention time:2.17 min (Method F). LCMS: 880.4 MH⁺.

Step 3.(1⁴S,16S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(2S,18S)-2,3,7,7-tetramethyl-4,10,15-trioxo-18-(pyridin-2-yldisulfaneyl)-16-oxa-8-thia-3,11,14-triazanonadecanoate

A solution of(1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-33,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-ylN-(4-((2-((2-aminoethyl)amino)-2-oxoethyl)thio)-4-methylpentanoyl)-N-methyl-L-alaninate(14 mg, 0.016 mmol) in DMF (0.2 mL) was added to solid (S)-4-nitrophenyl(2-(pyridin-2-yldisulfaneyl)propyl) carbonate (6.6 mg, 0.018 mmol).Catalytic HOAt and DIEA (10 mL, 0.057 mmol) were added to the resultantsolution and stirred at room temperature for 3 hours. The solution wasneutralized with acetic acid (10 mL) and applied to a reverse phasecolumn (RediSEP C18 (15.5 g)) and eluted with a gradient of acetonitrile(30% to 95%) in water with acetic acid (0.05%) to afford 18 mg (85%yield) of the title product. HPLC purity at 254 nm: 99%. Retention time:2.85 min (Method F). LCMS: 1129.4 MNa⁺.

Step 4. Synthesis of Compound 10

A solution of(1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-33,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(2S,18S)-2,3,7,7-tetramethyl-4,10,15-trioxo-18-(pyridin-2-yldisulfaneyl)-16-oxa-8-thia-3,11,14-triazanonadecanoate(17.7 mg, 0.00857 mmol) in DMF (1 mL) was treated with sodiumbicarbonate (1.8 mg, 0.0214 mmol) and water (50 mL). The resultantsolution was treated with peptide, Pv1 (31.5 mg, 0.0899 mmol) andstirred at room temperature for 3 hours, then applied to a reverse phasecolumn, RediSep C18 (15.5 g) and eluted with a gradient of acetonitrile(30% to 70%) in water with ammonium acetate (10 mM). The fractions werecombined, frozen and lyophilized to afford the product as a white solid,18.7 mg (50%). HPLC purity at 254 nm: 99%. Retention time: 6.49 min(Method G) LCMS: 2138.0 (M+2H)/2⁺, 1425.3 (M+3H)/3⁺.

Example 11: Synthesis of Compound 11

Step 1.(1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-85,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-ylN-(4-((4-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutyl)disulfaneyl)-4-methylpentanoyl)-N-methyl-L-alaninate

DM4 (10 mg, 0.013 mmol) and succinimidyl 4-(2-pyridyldithio)butanoate (6mg, 0.02 mmol) were mixed in DMF (0.26 mL). Triethylamine was added(0.015 mL) and the mixture was stirred for 2 h.1-(2-aminoethyl)-1H-pyrrole-2,5-dione hydrochloride (5 mg, 0.026 mmol)was added and after 3 h the mixture was directly loaded onto a RediSEPC18Aq (15.5 g) column and eluted with a gradient of acetonitrile (30% to95%) in water with acetic acid (0.05%) to afford 6 mg (40% yield) of thetitle product. HPLC purity at 254 nm: 92%. Retention time: 2.83 min(Method F). LCMS: 1020.4 MH⁺.

Step 2. Synthesis of Compound 11

A solution of(1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-33,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-ylN-(4-((4-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-4-oxobutyl)disulfaneyl)-4-methylpentanoyl)-N-methyl-L-alaninate(6 mg, 0.006 mmol) and Pv1 peptide (22.3 mg, 0.006 mmol) were dissolvedin DMF (0.12 mL) and treated with triethylamine (0.001 mL). After 30minutes the reaction mixture was directly loaded onto a RediSEP C8 (15.5g) column and eluted with a gradient of acetonitrile (35% to 75%) inwater with TFA (0.05%) to afford 16 mg (64% yield) of the titlecompound. HPLC purity at 254 nm: 98%. Retention time: 6.19 min (MethodG). LCMS: 2150.2 (M+2H)/2⁺, 1433.3 (M+3H)/3⁺.

Example 12. Synthesis of Compound 12

Step 1.((1⁴S,16S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-16,16,20,21-tetramethyl-10,19-dioxo-3,6-dioxa-14,15-dithia-9,20-diazadocosan-22-oate

DM4 (20 mg, 0.026 mmol) and succinimidyl 4-(2-pyridyldithio)butanoate(12 mg, 0.04 mmol) were mixed in DMF (0.75 mL). Triethylamine was added(0.045 mL) and the mixture was stirred for 2 h.1-(21-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-pyrrole-2,5-dionehydrochloride (9 mg, 0.036 mmol) was added and after 3 h the mixture wasdirectly loaded onto a RediSEP C18Aq (15.5 g) column and eluted with agradient of acetonitrile (30% to 95%) in water with acetic acid (0.05%)to afford 17 mg (61% yield). HPLC purity at 254 nm: 99%. Retention time:2.84 min (Method F). LCMS: 1108.4 MH⁺.

Step 2. Synthesis of Compound 12

A solution of(1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-33,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-16,16,20,21-tetramethyl-10,19-dioxo-3,6-dioxa-14,15-dithia-9,20-diazadocosan-22-oate(15 mg, 0.014 mmol) and Pv1 peptide (52 mg, 0.015 mmol) were dissolvedin DMF (0.28 mL) and treated with triethylamine (0.006 mL). After 30minutes the reaction mixture was directly loaded onto a RediSEP C8 (15.5g) column and eluted with a gradient of acetonitrile (35% to 60%) inwater with TFA (0.05%) to afford 25 mg (34% yield). HPLC purity at 254nm: 98%. Retention time: 6.26 min (Method G). LCMS: 2194.0 (M+2H)/2⁺,1463.0 (M+3H)/3⁺.

Example 13. Synthesis of Compound 13

Step 1. 1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-13,13,17,18-tetramethyl-7,16-dioxo-3-oxa-11,12-dithia-6,17-diazanonadecan-19-oate

DM4 (20 mg, 0.026 mmol) and succinimidyl 4-(2-pyridyldithio)butanoate(12 mg, 0.04 mmol) were mixed in DMF (0.75 mL). Triethylamine was added(0.045 mL) and the mixture was stirred for 2 h.1-(2-(2-aminoethoxy)ethyl)-1H-pyrrole-2,5-dione hydrochloride (5 mg,0.024 mmol) was added and after 3 h the mixture was directly loaded ontoa RediSEP C18Aq (15.5 g) column and eluted with a gradient ofacetonitrile (30% to 95%) in water with acetic acid (0.05%) to afford 13mg (41% yield) of the title product. HPLC purity at 254 nm: 94%.Retention time: 2.85 min (Method F). LCMS: 1064.4 MH⁺.

Step 2. Synthesis of Compound 13

A solution of(14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(S)-1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-16,16,20,21-tetramethyl-10,19-dioxo-3,6-dioxa-14,15-dithia-9,20-diazadocosan-22-oate(18 mg, 0.017 mmol) and Pv1 peptide (63 mg, 0.019 mmol) were dissolvedin DMF (0.34 mL) and treated with triethylamine (0.007 mL). After 30minutes the reaction mixture was directly loaded onto a RediSEP C8 (15.5g) column and eluted with a gradient of acetonitrile (35% to 60%) inwater with TFA (0.05%) to afford 25 mg (34% yield) of the titlecompound. HPLC purity at 254 nm: 99%. Retention time: 6.24 min (MethodG). LCMS: 2172.0 (M+2H)/2⁺, 1448.7 (M+3H)/3⁺.

Example 14. Synthesis of Compound 14

Step 1.(1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(S)-9,9,13,14-tetramethyl-1,6,12-trioxo-1-(((1S,2S)-2-(pyridin-2-yldisulfaneyl)cyclohexyl)oxy)-8-thia-2,5,13-triazapentadecan-15-oate

A solution of(1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-33,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-ylN-(4-((2-((2-aminoethyl)amino)-2-oxoethyl)thio)-4-methylpentanoyl)-N-methyl-L-alaninate(14 mg, 0.016 mmol; Example 10, Step 2) in DMF (0.2 mL) was added tosolid 4-nitrophenyl ((1S,2S)-2-(pyridin-2-yldisulfaneyl)cyclohexyl)carbonate (7.2 mg, 0.018 mmol). Catalytic HOAt and DIEA (10 mL, 0.057mmol) were added to the resultant solution and stirred at roomtemperature for 3 hours. The solution was neutralized with acetic acid(10 mL) and applied to a reverse phase column, RediSEP C18 (15.5 g) andeluted with a gradient of acetonitrile (30% to 95%) in water with aceticacid (0.05%) to afford 15 mg (80% yield) of the title compound. HPLCpurity at 254 nm: 98%. Retention time: 3.05 min (Method F). LCMS: 1147.4MH⁺.

Step 2. Synthesis of Compound 14

A solution of(1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-33,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(S)-9,9,13,14-tetramethyl-1,6,12-trioxo-1-(((1S,2S)-2-(pyridin-2-yldisulfaneyl)cyclohexyl)oxy)-8-thia-2,5,13-triazapentadecan-15-oate(17 mg, 0.015 mmol) and Pv1 peptide (47 mg, 0.013 mmol) were dissolvedin DMF (0.34 mL) and treated with triethylamine (0.007 mL). After 30minutes the reaction mixture was directly loaded onto a RediSEP C8 (15.5g) column and eluted with a gradient of acetonitrile (35% to 60%) inwater with TFA (0.05%) to afford 28 mg (37% yield). HPLC purity at 254nm: 99%. Retention time: 7.36 min (Method G). LCMS: 2158.0 (M+2H)/2⁺,1439.0 (M+3H)/3⁺.

Example 15. Synthesis of Compound 15

Step 1.(1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-85,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(S)-5,9,9,13,14-pentamethyl-6,12-dioxo-8-thia-2,5,13-triazapentadecan-15-oate

25 mg of(1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-33,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-ylN-(4-((2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)thio)-4-methylpentanoyl)-N-methyl-L-alaninate(0.027 mmol) and 40 mg ofN1-((3-methoxyphenyl)diphenylmethyl)-N1,N2-dimethylethane-1,2-diamine(0.11 mmol, 4 eq) were dissolved in dioxane (1 mL). After 3 h, thereaction appeared to be complete on LC/MS. 0.05 mL of TFA was added andthe mixture was loaded onto a 15.5 g C18Aq column and purified viastandard 5-100% B gradient (A: water w. 0.05% TFA; B: ACN w. 0.05% TFA).Product containing fractions were lyophilized to afford 22 mg of product(75% yield). HPLC purity at 254 nm: 98%. Retention time: 2.25 min(Method F). LCMS: 908.4 MH⁺.

Step 2.(1⁴S,1⁶S,3²S,33S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-85,14-dimethoxy-3³,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(S)-2,5,9,9,13,14-hexamethyl-1,6,12-trioxo-1-(((1S,2S)-2-(pyridin-2-yldisulfaneyl)cyclohexyl)oxy)-8-thia-2,5,13-triazapentadecan-15-oate

A solution of(1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-33,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(S)-5,9,9,13,14-pentamethyl-6,12-dioxo-8-thia-2,5,13-triazapentadecan-15-oate(15 mg, 0.016 mmol) in DMF (0.2 mL) was added to solid 4-nitrophenyl((1S,2S)-2-(pyridin-2-yldisulfaneyl)cyclohexyl) carbonate (6.6 mg, 0.018mmol). Catalytic HOAt and DIEA (10 mL, 0.057 mmol) were added to theresultant solution and stirred at room temperature for 3 hours. Thesolution was neutralized with acetic acid (10 mL) and applied to areverse phase column, RediSEP C18 (15.5 g) and eluted with a gradient ofacetonitrile (30% to 95%) in water with acetic acid (0.05%) to afford 16mg (82% yield) of the title product. HPLC purity at 254 nm: 97%.Retention time: 3.36 min (Method F). LCMS: 1175.5 MH⁺.

Step 3. Synthesis of Compound 15

A solution of(1⁴S,1⁶S,3²S,3³S,2R,4S,10E,12E,14R)-8⁶-chloro-1⁴-hydroxy-8⁵,14-dimethoxy-33,2,7,10-tetramethyl-1²,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(S)-2,5,9,9,13,14-hexamethyl-1,6,12-trioxo-1-(((1S,2S)-2-(pyridin-2-yldisulfaneyl)cyclohexyl)oxy)-8-thia-2,5,13-triazapentadecan-15-oate(16 mg, 0.014 mmol) and Pv1 peptide (52 mg, 0.015 mmol) were dissolvedin DMF (0.28 mL) and treated with triethylamine (0.008 mL). After 30minutes the reaction mixture was directly loaded onto a RediSEP C8 (15.5g) column and eluted with a gradient of acetonitrile (35% to 60%) inwater with TFA (0.05%) to afford 32 mg (44% yield) of the titlecompound. HPLC purity at 254 nm: 99%. Retention time: 6.86 min (MethodG). LCMS: 2172.0 (M+2H)/2⁺, 1448.4 (M+3H)/3⁺.

Example 16. Synthesis of Compound 16

Step 1. (4-((5-nitropyridin-2-yl)disulfaneyl)phenyl)methanol

A solution of (4-mercaptophenyl)methanol (0.74 g, 4.83 mmol) in THE (10mL) was treated with 5-nitro-2-((4-nitrophenyl)disulfaneyl)pyridine (1.0g, 3.23 mmol). The resultant suspension was stirred at room temperaturefor 2 hours, and the solvent was evaporated in vacuo. The residue wasdissolved in DCM and applied to a RediSep silica gel column and elutedwith a gradient of ethyl acetate (10% to 60%) in hexanes to afford theproduct (0.499 g, 52% yield). HPLC purity at 254 nm: 90%. Retentiontime: 2.72 min (Method F). MS data, 295.1 (M+H)+. ¹HNMR(DMSO-d₆) δ 9.18(s, 1H), 8.58 (d of d, 1H), 8.02 (d, 1H), 7.56 (d, 2H), 7.34 (d, 2H),5.24 (t, 1H) and 4.47 (d, 2H).

Step 2. 4-nitrophenyl (4-((5-nitropyridin-2-yl)disulfaneyl)benzyl)carbonate

A solution of 4-nitrophenyl chloroformate (255 mg, 1.26 mmol) in THE (5mL) was cooled on an ice-bath and treated with a solution(4-((5-nitropyridin-2-yl)disulfaneyl)phenyl)methanol (220 mg, 0.748mmol), triethyl amine (0.7 mL, 5.03 mmol), and 4-dimethyaminopyridine(45 mg, 0.368 mmol) in THE (5 mL) added over about 15 minutes. Theice-bath was removed, and the solution was stirred at room temperaturefor one hour and stored in a freezer overnight. The solvent wasevaporated in vacuo, and the residue was dissolved in DCM, applied to aRediSep silica gel column (12 g) and eluted with a gradient of ethylacetate (2% to 100%) in hexanes. The product was purified further byreverse phase chromatography on a RediSep C18 cartridge (50 g) elutedwith a gradient of acetonitrile (30% to 95%) in water with acetic acid(0.05%), to afford the product, 55 mg (16%). HPLC purity at 254nm: >99%. Retention time: 3.77 min (Method F). MS data, 460.7 (M+H).¹HNMR (CDCl₃) d 9.29 (d, 1H), 8.39 (d of d, 1H), 8.28 (d of d, 2H), 7.83(d of d, 1H), 7.55 (d of d, 2H), 7.44 (d of d), 7.36 (d of d, 2H) and5.26 (d, 2H).

Step 3.(14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(S)-11,11,15,16-tetramethyl-1-(4-((5-nitropyridin-2-yl)disulfaneyl)phenyl)-3,8,14-trioxo-2-oxa-10-thia-4,7,15-triazaheptadecan-7-oate

A solution of(14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-ylN-(4-((2-((2-aminoethyl)amino)-2-oxoethyl)thio)-4-methylpentanoyl)-N-methyl-L-alaninate(15 mg, 0.017 mmol; Example 10, Step 2) in DMF (1 mL) was added to solid4-nitrophenyl (4-((5-nitropyridin-2-yl)disulfaneyl)benzyl) carbonate (26mg, 0.0566 mmol). Catalytic HOAt and DIEA (10 mL, 0.057 mmol) were addedto the resultant solution and stirred at room temperature for 3 hours.The solution was neutralized with acetic acid (7 mL, 0.122 mmol) andapplied to a reverse phase column, RediSEP C18 (15.5 g) and eluted witha gradient of acetonitrile (30% to 95%) in water with acetic acid(0.05%). Further purification on a silica gel column, RediSep (4 g),using a gradient of methanol (0.2% to 6%) in DCM as the eluant, affordedthe title product (10.3 mg, 50% yield). HPLC purity at 254 nm: >99%.Retention time: 3.16 min (Method F). MS data, 1182.3 (M+H-H₂O)⁺, 1201.3(M+H)⁺, 1222.3 (M+Na)⁺.

Step 4. Synthesis of Compound 16

A solution of(14S,16S,32S,33S,2R,4S,10E,12E,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl(S)-11,11,15,16-tetramethyl-1-(4-((5-nitropyridin-2-yl)disulfaneyl)phenyl)-3,8,14-trioxo-2-oxa-10-thia-4,7,15-triazaheptadecan-17-oate(10.3 mg, 0.00857 mmol) in DMF (1 mL) was treated with sodiumbicarbonate (1.8 mg, 0.0214 mmol) and water (50 mL). The resultantsolution was treated with peptide, Pv1 (31.5 mg, 0.0899 mmol) andstirred at room temperature for 3 hours, then applied to a reverse phasecolumn, RediSep C18 (15.5 g) and eluted with a gradient of acetonitrile(30% to 70%) in water with ammonium acetate (10 mM). The fractions werecombined, frozen and lyophilized to afford the product as a white solid,18.7 mg (50%). HPLC purity at 254 nm: 99%. Retention time: 6.63 min(Method G). MS data, 2162.4 (M+2H)/2⁺, 1441.8 (M+3H)/3⁺, 1082.6(M+4H)/4⁺, 1435.7 (M+3H-H₂O)/3⁺.

Example A. Growth Delay Assay

Cells were plated in 96 well black walled-clear bottom plates (Griener),DLD-1 WT cells at 2500 cells per well, FaDu, and HeLa cells at 5000cells per well, and HCT116 at 3000 cells per well, in growth mediacontaining 10% FBS. Cells were allowed to adhere at room temperature for60 minutes before returning to a 37 C, 5% CO₂ incubator. After 24 hours,media was removed and replaced with fresh growth media containingvarious drug concentrations. Each drug concentration was added intriplicate. Non-drug treated controls contained growth media only. Cellswere returned to the incubator. Ninety-six hours after addition of drug,cells were fixed with 4% paraformaldehyde for 20 minutes and stainedwith Hoechst at 1 μg/mL. The plates were imaged on a Cytation 5 autoimager (BioTek) and cells were counted using CellProfiler(http://cellprofiler.org). The percent cell growth delay was calculatedand data plotted using GraphPad Prism

TABLE 5 Growth Delay Assay data DLD-1 HCT116 FaDu HeLa Example (IC₅₀,nM) (IC₅₀, nM) (IC₅₀, nM) (IC₅₀, nM) R²SH-1 9.8 4.3 2.8 2.6 (see Table2) R²SH-2 0.45 0.20 0.13 0.02 (see Table 2) 1 60.5 21.8 10.4 7.4 2 11421.1 15.4 8.0 3 45.1 19.3 10.1 6.9 4 11.7 2.5 1.4 0.85 5 9.4 2.7 2.30.95 6 8.3 3.3 5.2 1.8 7 NC* 3.4 4.6 2.0 8 9.8 3.0 3.4 1.6 NC* = Notcalculated

Example B: Effect on In Vitro Tubulin Polymerization

A fluorescence-based tubulin polymerization assay (Cytoskeleton Cat#BK011P) was performed to quantitate the impact of unconjugated DM4 andCompound 5 on in vitro tubulin polymerization. DM4 and Compound 5 wereprepared as 10 mM stocks in DMSO then diluted at 10× to 200, 50 and 5 μMin ultrapure distilled water for a final DMSO concentration of 0.2%. Kitreagents were defrosted rapidly then kept cold on ice to preventpremature polymerization. A tubulin reaction mixture was prepared on iceby mixing purified porcine brain tubulin, GTP, and glycerol buffer allin 1× kit buffer for final concentrations of 2 mg/mL tubulin, 1 mM GTPand 15% glycerol. 5 μL of DM4, Compound 5 or DMSO control were added toa pre-warmed black, half-well reaction plate at 37° C. for no longerthan 1 minute, to warm, but not allow for evaporation. 50 μL of tubulinreaction mixture was rapidly added to each well and immediately placedin a pre-warmed Cytation 5 imaging reader (BioTek). A kinetic readingwas performed at 360 excitation/450 emission for 2 hours at 37° C., withreadings every 2.5 minutes to follow the enhancement of fluorescence dueto incorporation of a fluorescent reporter into microtubules aspolymerization occurs.

FIG. 1 shows a plot of the effect of free DM4 and Compound 5 on in vitroβ-tubulin polymerization (in terms of relative fluorescence units) at0.5 μM, 5 μM, and 20 μM.

Example C: Kinetic Analysis of Conjugate Binding

Binding experiments were performed using a Biacore S200 instrument. ASeries S sensor chip with pre-immobilized streptavidin was conditionedwith 1 M NaCl in 50 mM NaOH. Biotin-labeled human tubulin derived fromHeLa cells was immobilized to the sensor chip at a concentration of 125μg/mL in HBS-P+ buffer at a flow rate of 10 l/min. A final 3000 RU(response units) of protein was directly immobilized to the chip. Aftertubulin immobilization, the sensor chip was washed with 50% isopropanol,50 mM NaOH and 1 M NaCl and subsequently allowed to equilibrate in assaybuffer for 4 hours. A streptavidin-biotin capture blank (reference FC)was used to monitor non-specific binding.

To collect kinetic binding data, Compound 5 diluted in assay buffer wasinjected over the flow cells at concentrations ranging from 100 M to0.048 M and 50 M to 0.024 M, at a flow rate of 60 L/minute and atemperature of 25° C. The complex was allowed to dissociate for 60seconds. Binding of compound to tubulin was monitored in real time toobtain on (Ka) and off (Koff) rates. The affinity constant (KD) wascalculated by steady state kinetics.

FIG. 2 depicts the kinetic analysis of Compound 5 binding to β-tubulinin vitro as determined by Biacore surface plasmon resonance. Compound 5is able to bind to 0-tubulin with a similar KD as free DM4 (3.55 μM) andslower on/off rates relative to free DM4.

Example D: Efficacy of Compound 5 in a Mouse Colorectal Cancer Model

Six-week-old female athymic nude Foxn^(nu) mice were obtained fromTaconic Labs (Cat #NCRNU-F) and were housed 5 per cage on Alpha-Dribedding in a disposable caging system. Human HCT116 cells derived fromcolorectal carcinoma were diluted 1:1 in Phenol Red-free Matrigel andsubcutaneously implanted into the left flank of each mouse at a densityof 2.5×10⁶ cells in 100 μL. When xenografts reached a mean volume of100-200 mm³, mice were randomized into groups and treated as detailed inthe table below. Mice were administered intraperitoneal (IP) doses ofvehicle or 0.21, 0.29, 0.35, 0.42 μmole/kg Compound 5 (equivalent to1.1, 1.4, 1,7, or 2 mg/kg Compound 5) or 0.42 μmole/kg unconjugated DM4(equivalent to 0.33 mg/kg unconjugated DM4). Doses were prepared bydiluting 0.1 mg/μL DMSO stocks in 5% mannitol in citrate buffer and wereadministered QDX4 with a two day interval between the second and thirddoses, at a volume of 12 mL/kg (300 μL per 25 g mouse). Xenograft tumorswere measured by calipers and volume was calculated using the equationfor ellipsoid volume: Volume=π/6×(length)×(width)². Body weight ofanimals was measured at the same time as tumor volume assessment.Animals were removed from the study due to death, tumor size exceeding2000 mm³ or loss of >20% body weight. Kaplan-Meier analysis was used toevaluate survival rate based on death or removal from study.

FIG. 3A shows a plot of the mean tumor volume in nude mice bearingHCT116 colorectal flank tumors dosed with DM4 or Compound 5.

FIG. 3B shows the percent change in body weight of nude mice bearingHCT116 colorectal flank tumors dosed with DM4 or Compound 5 relative today 0.

FIG. 4 depicts a Kaplan-Meier plot of nude mice bearing HCT116colorectal flank tumors dosed with DM4 or Compound 5. Animals wereremoved from the study due to either death, tumor size exceeding 2000mm³ or due to loss of greater than 20% body weight. Free DM4 induced thespontaneous death of half of the DM4 group of animals during thepost-dosing period. As shown in FIG. 4 , Compound 5 safely deliversamounts of DM4 in vivo that otherwise result in systemic toxicity anddeath when dosed as free DM4.

Example E: Effect of Compound 6 on Lung Metastases in a Mouse LungCancer Model

Mouse 4T1-iRFP cancer cells derived from mouse mammary carcinoma andtransfected with near infrared fluorescent protein (iRFP) were culturedas a monolayer at 37° C. in a humidified atmosphere with 5% CO₂. Cellswere passaged between one and three days prior to implantation and mediawas replaced every 2-3 days as needed to maintain cell viability. Cellswere not allowed to exceed 80% confluency. On the day of implantation,cells were trypsinized, washed with complete media and pelleted bycentrifugation at 1200 rpm for 5 minutes. The supernatant was decanted,and cells were washed three times with sterile PBS and pelleted bycentrifugation. During the final centrifugation, viability wasdetermined using trypan blue exclusion. Cells were resuspended insterile PBS a final concentration of 5×10⁵ cells/100 μL. Cells weredrawn into sterile 1 cc tuberculin syringes with a 27-gauge needle. Airbubbles were removed, and excess cell mixture was expelled back into theconical tube leaving an injection volume of 100 μL in each syringe. The100 L of cells were injected directly into the medial tail vein ofsix-week-old female athymic nude Foxn^(nu) mice (Taconic Labs Cat#NCRNU-F).

Three days after cell injection, mice were administered intraperitonealdoses of vehicle or 2.5 mg/kg Compound 6 once daily for 2 days followedby 2 days of no treatment, followed by a single dose of Compound 6, fora total of three total doses of Compound 6. Eleven days after injection,mice were euthanized, and the lungs were removed for imaging using theLI-COR PEARL Trilogy small animal imager to visualize and quantitatelung metastasis and evaluate compound effect on tumor growth.

FIG. 5A depicts the ventral view and extracted lungs of nude miceinoculated with 4T1-RFP fluorescent cells via tail vein injection andimaged 11 days after inoculation and after 3 doses of vehicle orCompound 6.

FIG. 5B depicts a graph of the fluorescent signal from extracted lungsof 4T1-RFP inoculated mice after 3 doses of vehicle or Compound 6.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference, including withoutlimitation all patent, patent applications, and publications, cited inthe present application is incorporated herein by reference in itsentirety.

What is claimed is:
 1. A compound of Formula (I):R²—L—R¹  (I) or a pharmaceutically acceptable salt thereof, wherein: R¹is a peptide capable of selectively delivering R²L- across a cellmembrane having an acidic or hypoxic mantle; R² is selected from thegroup consisting of:

L is the following group:

wherein R³, R⁴, R⁵, and R⁶, are each independently selected from H, C₁₋₄alkyl, C₁₋₄ alkenyl, C₆₋₁₀ aryl, C₃₋₁₀ cycloalkyl, 5-10 memberedheteroaryl, 4-10 membered heterocycloalkyl, halo, CN, NO₂, OR^(a1),SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1),OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1), wherein said C₁₋₄alkyl, C₁₋₄ alkenyl, C₆₋₁₀ aryl, and 5-10 membered heteroaryl are eachoptionally substituted with 1, 2, or 3 substituents independentlyselected from halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1); or R³ and R⁴ together with the carbon atom towhich they are attached form a C₃₋₁₄ cycloalkyl group or 4-14 memberedheterocycloalkyl group, each optionally substituted with 1, 2, or 3substituents independently selected from C₁₋₄ alkyl, halo, CN, NO₂,OR^(a1), SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1),OC(O)R^(b1), OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1); or R³ and R⁵ togetherwith the carbon atoms to which they are attached form a C₃₋₁₄ cycloalkylgroup or 4-14 membered heterocycloalkyl group, each optionallysubstituted with 1, 2, or 3 substituents independently selected fromC₁₋₄ alkyl, halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1); or R⁴ and R⁶ together with the carbon atoms towhich they are attached form a C₃₋₁₄ cycloalkyl group or 4-14 memberedheterocycloalkyl group, each optionally substituted with 1, 2, or 3substituents independently selected from C1-4 alkyl, halo, CN, NO₂,OR^(a1), SR^(a1), C(O)R^(b1), C(O)NR^(c1)R^(d1), C(O)OR^(a1),OC(O)R^(b1), OC(O)NR^(c1)R^(d1), NR^(c1)R^(d1), NR^(c1)C(O)R^(b1),NR^(c1)C(O)OR^(a1), and NR^(c1)C(O)NR^(c1)R^(d1); or R⁵ and R⁶ togetherwith the carbon atom to which they are attached form a C₃₋₁₄ cycloalkylgroup or 4-14 membered heterocycloalkyl group, each optionallysubstituted with 1, 2, or 3 substituents independently selected fromC₁₋₄ alkyl, halo, CN, NO₂, OR^(a1), SR^(a1), C(O)R^(b1),C(O)NR^(c1)R^(d1), C(O)OR^(a1), OC(O)R^(b1), OC(O)NR^(c1)R^(d1),NR^(c1)R^(d1), NR^(c1)C(O)R^(b1), NR^(c1)C(O)OR^(a1), andNR^(c1)C(O)NR^(c1)R^(d1); A is H or C₁₋₄ alkyl; and R^(a1), R^(b1),R^(c1), and R^(d1) are each independently selected from H, C₁₋₆ alkyl,C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ haloalkyl, OH, CN, NO₂, and CO₂CH₃;wherein said C₁₋₆ alkyl and C₂₋₆ alkenyl are each optionally substitutedwith OH, CN, NO₂, or CO₂CH.
 2. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R¹ is a peptidecapable of selectively delivering R²L- across a cell membrane having anacidic or hypoxic mantle having a pH less than about 6.0.
 3. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R¹ is a peptide comprising at least one of the followingsequences: (SEQ ID NO. 1; Pv1) ADDQNPWRAYLDLLFPTDTLLLDLLWCG,(SEQ ID NO. 2; Pv2) AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG, and(SEQ ID NO. 3; Pv3) ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG; and(SEQ ID No. 6; Pv6) AAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG;

wherein R¹ is attached to L through a cysteine residue of R¹.
 4. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R¹ is a peptide comprising at least the following sequence:(SEQ ID NO. 1; Pv1) ADDQNPWRAYLDLLFPTDTLLLDLLWCG.


5. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R¹ is a peptide comprising at least the followingsequence: (SEQ ID NO. 2; Pv2) AEQNPIYWARYADWLFTTPLLLLDLALLVDADECG.


6. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R¹ is a peptide comprising at least the followingsequence: (SEQ ID NO. 3; Pv3) ADDQNPWRAYLDLLFPTDTLLLDLLWDADECG.


7. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R¹ is a peptide comprising at least the followingsequence: (SEQ ID NO. 6; Pv6) AAEQNPIYWWARYADWLFTTPLLLLDLALLVDADEGTCG.


8. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R² is:


9. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R² is:


10. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R² is:


11. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R² is:


12. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein L is:


13. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R³, R⁴, R⁵, and R⁶ are each independently selected fromH and C₁₋₄ alkyl.
 14. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein R³, R⁴, R⁵, and R⁶ are each H.
 15. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein A is H.
 16. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein A is CH₃.
 17. The compound of claim 1,selected from:

or a pharmaceutically acceptable salt of any of the aforementioned,wherein Pv1 is a peptide comprising at least the following sequence:ADDQNPWRAYLDLLFPTDTLLLDLLWCG (SEQ ID NO. 1).
 18. A pharmaceuticalcomposition that comprises a compound of claim 1, or a pharmaceuticallyacceptable salt thereof.
 19. The compound of claim 1, which is:

or a pharmaceutically acceptable salt thereof, wherein Pv1 is a peptidecomprising at least the following sequence: ADDQNPWRAYLDLLFPTDTLLLDLLWCG(SEQ ID NO. 1).
 20. The compound of claim 1, which is:

or a pharmaceutically acceptable salt thereof, wherein Pv1 is a peptidecomprising at least the following sequence: ADDQNPWRAYLDLLFPTDTLLLDLLWCG(SEQ ID NO. 1).
 21. The compound of claim 1, which is:

or a pharmaceutically acceptable salt thereof, wherein Pv1 is a peptidecomprising at least the following sequence: ADDQNPWRAYLDLLFPTDTLLLDLLWCG(SEQ ID NO. 1).